Methods for the site-selective introduction of halogen into natural products

ABSTRACT

Organohalides (e.g., organofluorides) represent a rapidly expanding proportion of molecules used in pharmaceuticals, diagnostics, agrochemicals, and materials. The present invention exploits the naturally occurring acetate condensation pathway as a means of introducing halogenated building blocks into natural product scaffolds. In an exemplary embodiment, the invention provides a pathway involving at least one polyketide synthase system and utilizes haloacetate or a haloacetate synthon (e.g., halomalonate) to incorporate an organohalide into the polyketide backbone in vitro. In an exemplary embodiment, the invention provides an analogous method for site-selective introduction of haloacetate or a synthon into polyketide products in vivo. The present invention expands the scope of existing chemical methods for producing halogenated natural product scaffolds such as complex halogenated natural products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims, under 35 USC 119(e), the benefit of U.S.Provisional Application No. 61/868,494 filed Aug. 21, 2013, which isincorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.1-DP2-OD008696 awarded by National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

Reagents and methods for the in vitro and in vivo synthesis ofpolyketides are provided.

BACKGROUND OF THE INVENTION

The catalytic diversity of biological systems provides enormouspotential for application of living cells to the scalable production ofpharmaceuticals, fuels, and materials (Ro, et al., Nature, 440: 940-943(2006); Atsumi, et al., Nature 451, 86-89 (2008); Cane, et al., Science282:63-68 (1998); and Weeks, et al., Biochemistry 50, 5404-5418 (2011)).However, the scope of innovation of living organisms is typicallylimited to functions that confer a direct advantage for cell growth,thereby maximizing biomass as the end product rather than a distinctmolecule or reaction of interest. In contrast, synthetic biologyapproaches allow us to disconnect some of these remarkable biochemicaltransformations from cell survival and reconnect them differently forthe targeted synthesis of alternative classes of compounds. Oneparticularly interesting area of opportunity is the development ofmethods to introduce halides into complex small molecule scaffolds,which has become a powerful strategy for the design of syntheticpharmaceuticals. Indeed, it is estimated that 20-30% of drugs, includingmany of the top sellers, contain at least one fluorine atom (Müller, etal., Science, 317:1881-1886 (2007); D. O'Hagan, Chem. Soc. Rev.37:308-319 (2008); and Furuya, et al., Nature, 473:470-477 (2011)). Forexample, tecent innovations have expanded the scope of synthetic CF bondforming methodologies, but the unusual elemental properties of fluorinethat serve as the basis for its success also continue to restrict therange of molecular structures that can be accessed (Ball, et al., J. Am.Chem. Soc., 131:3796-3797 (2009); Watson et al., Science, 325:1661-1664(2009); Rauniyar, et al., Science, 334:1681-1684 (2011); and Lee, etal., Science, 334:639-642 (2011)). As such, the invention of alternativeroutes for the site-selective introduction of halogens into structurallydiverse molecules, particularly under mild conditions, remains anoutstanding challenge.

In comparison to synthetic small molecules, halogens, e.g., fluorine,have limited distribution in naturally occurring organic compounds. Forexample, the only organofluorine natural products characterized to dateconsist of a small set of simple molecules associated with thefluoroacetate pathway of Streptomyces cattleya, a soil bacterium thathouses the remarkable ability to catalyze the formation of CF bonds fromaqueous fluoride (FIG. 1A) (Dong, et al., Nature, 427:561-565 (2004);and D. O'Hagan, J. Fluorine Chem., 127:1479-1483 (2006)).

The backbones of several large classes of medicinally-relevant naturalproducts including polyketides, isoprenoids, steroids, alkaloids,eicosanoids, leukotrienes, and others are biosynthesized directly fromthe assembly and tailoring of simple acetate units (FIG. 1A).Introduction of the haloacetate monomer in place of acetate would allowincorporation of fluorine into the backbone of these targets and createnew molecular function by combining the medicinal chemistry advantagesof fluorine with the structural complexity and bioactivity of naturalproducts. The present invention provides a method for accomplishing thisgoal.

BRIEF SUMMARY OF THE INVENTION

In various embodiments, the present invention is directed towardincreasing the typically low yields associated with conventionalsynthesis of halo-polyketides and other natural products (e.g.,isoprenoids, steroids, alkaloids, eicosanoids, leukotrienes) formed bythe condensation of acetate groups. In various embodiments, the currentinvention is directed toward new methods for the synthesis of naturalproducts such as functionalized triketides. In various embodiments,malonyl-CoA is an example of a universal extender in triketidesynthesis, and the synthesis of other natural products involvingcondensation of acetate.

Amongst various embodiments, the present invention provides direct andefficient access to halomalonyl-CoA, a halogenated analog of one ofnature's most ubiquitous carbon nucleophiles. Also provided are methodsof using this compound as an extender unit, which affords a generalmethod for direct incorporation of halogen into a large array ofpolyketide and other natural product structures.

It is a further object of the invention to provide a host microorganism,wherein a heterologous nucleic acid sequence comprises a DNA fragmentcoding for an enzyme competent to incorporate a halogenated malonyl-CoAanalogue in a synthetic pathyway for a polyketide.

It is still a further object of the invention to provide a methodwherein the host microorganism is a prokaryote.

It is an additional object of the invention to provide a method whereinthe prokaryote is Escherichia coli.

Is it still another object of the invention to provide a method forsynthesizing a halogenated ketide, e.g., a polyketide, in a hostmicroorganism, wherein the method comprises introducing into the hostmicroorganism a compound of Formula I, II and/or III and at least oneheterologous nucleic acid sequence. Exemplary heterologous nucleic acidsequences encode for an enzyme that catalyzes the ligation of CoA ontohalomalonic acid, haloacetic acid or the condensation of one of thesehaloacids on to a substrate for a polyketide.

It is still a further object of the invention to provide DNA fragments,expression vectors, and host cells for carrying out the methodsdescribed herein.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description that follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned through routine experimentation uponpractice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Synthetic biology of fluorine. (A) The fluoroacetate pathwayand its metabolites represent the known scope of biological fluorinechemistry, starting with fluoride and S-adenosylmethionine, to producefluoroacetate and fluorothreonine as the end products (right to left,grey box). This scope is greatly expanded by engineering downstreampathways to use fluoroacetate as a building block for introduction offluorine site-selectively into large families of natural productsconstructed from acetate backbones (left to right, red box). Red dotsrepresent positions that can be fluorinated by incorporation of afluoroacetate monomer without altering the carbon skeleton, includinglocations where fluorine would replace a methyl group derived frompropionate or where downstream tailoring steps have occurred on thefinal structure. (B) Assembly of acetate units in the biosynthesis ofpolyketide natural products.

FIG. 2A-B. Enzymatic production of activated extender units for CC bondformation reactions. (A) Formation of malonyl-CoA (left) andfluoromalonyl-CoA (right) from 500 μM CoA and either acetate orfluoroacetate, respectively (AckA, acetate kinase; Pta,phosphotransacetylase; ACCase, acetyl-CoA carboxylase). Values arereported as the mean±s.d. (n=3). (B) Kinetic parameters for malonateactivation (MatB, malonyl-CoA synthetase). Kinetic parameters arereported as mean±s.e. (n=3) as determined from non-linear curve-fitting.Error in the k_(cat)/K_(M) parameter was obtained from propagation oferror from the individual kinetic terms.

FIG. 3A-B. A chain extension and ketoreduction cycle with a fluorinatedextender using a simple polyketide synthase, NphT7. (A) Reactionscatalyzed by NphT7 and PhaB. (B) Steady-state kinetic parameters forNphT7-catalyzed C—C bond formation measured using a coupled assay withPhaB. Data points are reported as the mean±s.d. (n=3). Kineticparameters are reported as mean±s.e. (n=3) as determined from non-linearcurve-fitting. Error in the k_(cat)/K_(M) parameter was obtained frompropagation of error from the individual kinetic terms.

FIG. 4A-D. Production of fluorinated polyketides in vitro and in vivo byDEBS_(Mod6)+TE. (A) Reaction catalyzed by DEBS_(Mod6)+TE using theNDK-SNAC substrate with various extender units (NDK-SNAC, nativediketide N-acetylcysteamine thioester,(2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester). (B)Chain extension by DEBS_(Mod6)+TE to form triketide lactones monitoredby LC-MS (TKL, m/z=169; F-TKL, m/z=173). CoA, ATP, and ATP regenerationsystem are included in all in vitro reactions. Data are normalized withrespect to the TKL peak. (C) Selectivity of DEBS_(Mod6)+TE andDEBS_(Mod3)+TE for methylmalonyl-CoA vs. fluoromalonyl-CoA extender unitas monitored by TKL (m/z=169) and F-TKL (m/z=173) formation. Conditionsinclude wild-type modules, AT⁰ modules, and AT⁰ modules in conjunctionwith the trans-AT from the disorazole PKS (DszsAT). Values are reportedas the mean±s.d. (n=3). (KR*, the KR domain of Mod3 is inactive). (D)LC-MS traces showing regioselective tetraketide lactone formation usingthe DEBS mini-PKS consisting of DEBS_(Mod2) and DEBS_(Mod3)+TE (Me/Me,2-methyl-4-methyl-tetraketide lactone, m/z=227; Me/F,2-fluoro-4-methyl-tetraketide lactone, m/z=231; F/Me,2-methyl-4-fluoro-tetraketide lactone, m/z=231). Me/Me was producedusing DEBS_(Mod2)/DEBS_(Mod3)+TE and methylmalonate (1). Me/F wasproduced using DEBS_(Mod2)/DEBS_(Mod3)AT⁰+TE, DszsAT, methylmalonyl-CoA,and fluoromalonate (2). F/Me was produced usingDEBS_(Mod2)AT⁰/DEBS_(Mod3)+TE, methylmalonyl-CoA, and fluoromalonate(3). Data are normalized with respect to the Me/Me peak. All reactionscontained MatB and the ATP regeneration system.

FIG. 5A-B. SDS-PAGE gels of purified proteins. (A) Enzymes used ingeneration of malonyl-CoA extender units (1, AckA; 2, Pta; 3, AccA; 4,AccB; 5, AccC; 6, AccD; 7, MatB; 8, Epi). (B) Enzymes used for chainextension reactions (9, THNS; 10, NphT7). (C) Enzymes used forpolyketide production (11, DEBS_(Mod2); 12, DEBS_(Mod2)/AT⁰; 13,DEBS_(Mod3)+TE; 14, DEBS_(Mod3)/ATQFTE; 15, DEBS_(Mod6)+TE; 16,DEBS_(Mod6)/AT⁰+TE; 17, DszsAT).

FIG. 6A-C. Formation of fluoroacetyl-CoA using AckAPta. (A) HPLCchromatograms monitoring fluoroacetyl-CoA formation by A_(260 nm). (B)Plot of the conversion of free CoA to fluoroacetyl-CoA. (C) Kineticparameters for AckA and Pta measured using spectrophotometric assays.(Walker, et al., ACS Chem. Biol. (2012)). Values are reported as themean±s.e. as determined from nonlinear curve fitting.

FIG. 7A-C. Steady state kinetic analysis of MatB. (A) Malonate. (B)Methylmalonate. (C) Fluoromalonate. Values are reported as the mean±s.eas determined from nonlinear curve fitting.

FIG. 8A-C. NMR spectra of enzymatically synthesized fluoromalonyl-CoA.(A) ¹H NMR. (B) ¹³C NMR. (C) ¹⁹F NMR. Spectra reflect partial (¹H, ¹⁹F)or complete (¹³C) H-D exchange at the fluorinated position based onincubation time.

FIG. 9. Efficiency of polyketide production with tetrahydroxynaphthalenesynthase (THNS) using different extender regeneration systems. THNS usesonly malonyl-CoA as both starter and extender unit (Kumar, et al.,Method. Enzymol., 269-293 (2004)). All samples contained a fixed amountof malonyl- or acetyl-CoA (0.5 mM), and relative THN production wasmonitored at A_(510 nm). Samples with no regeneration system (1, 2) werecompared to those containing regeneration systems related tonon-productive decarboxylation (3, 4) and hydrolysis (5), while alsoproviding additional substrate (5-9) in situ. Values are reported as themean±s.d. (n=3).

FIG. 10. Structural alignment of NphT7 and the DEBS_(Mod5) ketosynthase(KS) domain. The NphT7 structure was predicted using Phyre2 (Kelley, etal., Nat. Prot., 4:363-371 (2009)) and based on a type III3-oxoacyl-(acyl-carrier protein) synthase from Burkholderia xenovorans(PDB ID 4EFI). Despite low sequence identity (<20%), the predictedstructure overlays well with the KS domain from DEBS_(Mod5) (Tang, etal., Proc. Nat. Ac. Sc U.S.A., 103:11124-11129 (2006)). Active siteresidues (C119, H334 H374 (N in NphT7); DEBS numbering) are highlighted.

FIG. 11. Characterization of enzymatically synthesized2-fluoro-3-hydroxybutyryl-CoA. (A) LC/MS trace of2-fluoro-3-hydroxybutyryl-CoA isolated from enzymatic reaction mixtures(m/z 872). (B) ¹⁹F NMR spectrum indicates that both diastereomers areproduced. (C) ¹H-¹⁹F HMBC in D₂O. Based on data from other-fluoroalcohols (Mohanta, et al., J. Am. Chem. Soc., 127:11896-11897(2005)), the ¹⁹F resonance for the anti configuration of the fluorineand hydroxyl groups should be found upfield of the syn and was assignedas the major product. If PhaB maintains is native selectivity as anR-specific acetoacetyl-CoA reductase, the anti product is(2S,3R)-2-fluoro-3-hydroxybutyryl-CoA.

FIG. 12. Amplification of TKL formation using MatB. All reactionscontained 400 mM sodium phosphate, pH 7.5, phosphoenolpyruvate (50 mM),TCEP (5 mM), magnesium chloride (10 mM), ATP (2.5 mM), pyruvate kinase(27 U/mL), myokinase (10 U/mL), methylmalonyl-CoA epimerase (5 μM),methylmalonate (20 mM), NDK-SNAC (1 mM) and DEBS_(Mod6)+TE (10 μM). Thesource of extender unit was either methylmalonyl-CoA (0.5-10 mM) or MatB(40 μM) and CoA (0.5 mM). (A) Dependence of TKL formation onmethylmalonyl-CoA. Data are average±s.d. (n=3). (B) Comparison of TKLyield with and without MatB regeneration. Values are reported as themean±s.d. (n=3).

FIG. 13A-C. Time-course for TKL and F-TKL formation by DEBS_(Mod6)+TEwith substrate regeneration. (A) LC/MS traces monitoring TKL formation(m/z 169) from 2.5 mM NDK-SNAC. (B) Plot of NDK-SNAC and TKLconcentrations. Initial rate: 1.5 min⁻¹ (C) LC/MS traces monitoringF-TKL formation (m/z 173) from 10 mM NDK-SNAC. (D) Plot of NDK-SNAC andF-TKL concentrations. Initial rate: 0.14 h⁻¹. (▪, NDK-SNAC; ▪, TKL; ▪,F-TKL).

FIG. 14A-C. 1D-NMR spectra of synthetic F-TKL standard in CDCl₃. (A) ¹HNMR. (B) ¹³C NMR. (C) ¹⁹F NMR. The relative keto:enol ratio in CDCl₃depends on concentration and increases with decreasing concentration.

FIG. 15A-D. 2D-NMR spectra of synthetic F-TKL standard in CDCl₃. (A)COSY. (B) ¹H-¹³C HSQC. (C) ¹H-¹³C HMBC. (D) ¹H-¹⁹F HMBC (see also FIG.16C).

FIG. 16A-C. Stereochemical analysis for F-TKL. (A) ¹H NOESY spectrum ofsynthetic F-TKL standard in CDCl₃. The same ratio between epimers isobserved for enzymatically produced F-TKL. (B) Molecular modelingresults for F-TKL. The lowest energy conformations of the two F-TKL ketodiastereomers were selected based on a conformational search(Macromodel) using Maestro 9.3 (Schrödinger, Inc). Only the 2S epimerwould be expected to show a single NOE coupling between H₂ and H₅, asobserved. (C) ¹H-¹⁹F HMBC of keto isomer region of synthetic F-TKLstandard in CDCl₃, showing crosspeaks for the major and minor epimers.

FIG. 17A-B. GC-MS and ¹⁹F NMR comparison of enzymatic F-TKL to theauthentic F-TKL synthetic standard. (A) Comparison of EI mass spectra ofthe standard (t_(R)=8.51 min) compared to the enzymatic product(t_(R)=8.56 min) (B) Comparison of ¹⁹F NMR spectra in CDCl₃. The ketoform is dominant at this concentration.

FIG. 18. Test for covalent inhibition of DEBS_(Mod6)+TE byfluoromalonyl-CoA. DEBS_(Mod6)+TE was incubated for 18 h in a F-TKL orTKL reaction. The enzyme was then isolated by Sephadex G-25 and testedfor its ability to produce TKL. Values are reported as the mean±s.d.(n=3).

FIG. 19. ¹⁹F NMR (90% H₂O, 10% D₂O, pH 7.5) of reaction mixture forF-TKL formation by DEBS_(Mod6)+TE and MatB. ¹⁹F NMR analysis of thereaction mixture indicates that the major pathway for loss offluoromalonyl-CoA appears to be hydrolysis rather than unproductivedecarboxylation. In addition, no detectable defluorination was observed.(IS, 5-fluorouracil, 50 μM).

FIG. 20. R-TKL production in vitro by DEBS_(Mod2)/AT⁰ under substrateregeneration conditions. When incubated overnight with NDK-SNAC (500 μM)and the methylmalonyl-CoA regenerating system, DEBS_(Mod2)/AT⁰ convertedall the NDK-SNAC to TKL. With the fluoromalonyl-CoA regeneration systemit consumed 82% of the NDK-SNAC with 43% conversion to F-TKL. With themalonyl-CoA regeneration system it consumed 100% of the NDK-SNAC with<0.01% conversion to H-TKL. Values are reported as the mean±s.d. (n=3).

FIG. 21. ESI-MS/MS data for tetraketide lactones. Authentic standardsare not available for the fluorinated compounds. Both fluorinatedtetraketide lactones exhibit a mass corresponding to the loss of HF (20amu). Fragments resulting from multiple losses were not assignedstructures; however, two of these fragments (a, b) present in thedimethyl tetraketide appear to be shifted by 4 amu in the2-fluoro-4-methyltetraketide lactone, suggesting a fluorine for methylsubstitution. Furthermore, two more fragments (c, d) appear to bepresent in the methyl form in one of the fluorinated tetraketidelactones and the fluoro form in the other regio-isomer. Thesefragmentation patterns suggest the 2-fluoro-4-methyl- and the2-methyl-4-fluoro tetraketide lactones are indeed distinctregio-isomers.

FIG. 22A-C. F-TKL production in vivo. (A) LC/MS traces showing F-TKLformation (m/z 173) in E. coli cell lysate. (B) LC/MS traces showingF-TKL formation (m/z 173) by E. coli cell culture upon feeding withNDK-SNAC. (C) In vivo selectivity data showing F-TKL production comparedto H-TKL and TKL in fluoromalonate-fed E. coli resting cells expressingeither DEBS_(Mod6)+TE or DEBS_(Mod6)+TE/AT⁰ and MatB. Bars representmean±s.d. (n=3) with individual samples marked (▪ ▴ ).

FIG. 23. Hydrolysis and regeneration reactions for F-TKL production byDEBS_(Mod6)+TE. Reaction scheme showing enzymes present in F-TKL formingreactions including observed non-productive hydrolysis reactions (red)and the ATP regenerating system (blue).

FIG. 24A-B. (A) Extracted ion LC/MS trace of enzymatic chloro-triketideproduct, m/z 189 (negative mode). (B) ESI mass spectrum of the peak inA. The expected isotopes are observed for 35Cl- and 37Cl-triketide.

FIG. 25. Organofluorine pharmaceuticals.

FIG. 26. Naturally-occurring organofluorines that have been identifiedto date are structurally simple but include highly toxic compounds suchas fluoroacetate and fluorocitrate. However, by developing newdownstream reactions to utilize fluoroacetate as a building block,fluorine could be incorporated site selectively into the backbones andside-chains of many large classes of modularly synthesized naturalproducts. (Fluorination sites indicated by a red dot).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides compounds, cells, systems and methodswhich expand the halogen chemistry of living systems using engineeredpathways to link simple biogenic organohalide building blocks into morecomplex halogenated small molecule targets. Because of the modularnature of the biosynthetic pathways used to produce polyketides andrelated acetate-derived natural products, the present invention opensthe door to general strategies for exploring the halogen syntheticbiology of complex natural products, and for producing such products.

Before the invention is described in greater detail, it is to beunderstood that the invention is not limited to particular embodimentsdescribed herein as such embodiments may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the invention describedherein is not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided might be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featureswhich may be readily separated from or combined with the features of anyof the other several embodiments without departing from the scope orspirit of the invention. Any recited method may be carried out in theorder of events recited or in any other order that is logicallypossible. Although any methods and materials similar or equivalent tothose described herein may also be used in the practice or testing ofthe invention, representative illustrative methods and materials are nowdescribed.

In describing the present invention, the following terms will beemployed, and are defined as indicated below.

II. Definitions

The terms “host microorganism” and “cell” are used interchangeablyherein to refer to a living biological cell that can be transformed viainsertion of an expression vector. Thus, a host organism or cell asdescribed herein may be a prokaryotic organism (e.g., an organism of thekingdom Eubacteria) or a eukaryotic cell. As will be appreciated by oneof ordinary skill in the art, a prokaryotic cell lacks a membrane-boundnucleus, while a eukaryotic cell has a membrane-bound nucleus. Apreferred prokaryotic cell is Escherichia coli. Preferred eukaryoticcells are those derived from fungal, insect, or mammalian cell lines.

The term “heterologous DNA” as used herein refers to a polymer ofnucleic acids wherein at least one of the following is true: (a) thesequence of nucleic acids is foreign to (i.e., not naturally found in) agiven host microorganism; (b) the sequence may be naturally found in agiven host microorganism, but in an unnatural (e.g., greater thanexpected) amount; or (c) the sequence of nucleic acids comprises two ormore subsequences that are not found in the same relationship to eachother in nature. For example, regarding instance (c), a heterologousnucleic acid sequence that is recombinantly produced will have two ormore sequences from unrelated genes arranged to make a new functionalnucleic acid. Specifically, the present invention describes theintroduction of an expression vector into a host microorganism, whereinthe expression vector contains a nucleic acid sequence coding for anenzyme that is not normally found in a host microorganism. Withreference to the host microorganism's genome, then, the nucleic acidsequence that codes for the enzyme is heterologous.

The term “polyketide synthase pathway” is used herein to refer to thepathway that utilizes acetyl-CoA or malonyl-CoA as extender subunits orextender subunit synthons in condensation reactions to form reactiveβ-keto units.

The terms “expression vector” or “vector” refer to a compound and/orcomposition that transduces, transforms, or infects a hostmicroorganism, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell, or in a manner not nativeto the cell. An “expression vector” contains a sequence of nucleic acids(ordinarily RNA or DNA) to be expressed by the host microorganism.Optionally, the expression vector also comprises materials to aid inachieving entry of the nucleic acid into the host microorganism, such asa virus, liposome, protein coating, or the like. The expression vectorscontemplated for use in the present invention include those into which anucleic acid sequence can be inserted, along with any preferred orrequired operational elements. Further, the expression vector must beone that can be transferred into a host microorganism and replicatedtherein. Preferred expression vectors are plasmids, particularly thosewith restriction sites that have been well documented and that containthe operational elements preferred or required for transcription of thenucleic acid sequence. Such plasmids, as well as other expressionvectors, are well known to those of ordinary skill in the art.

The term “transduce” as used herein refers to the transfer of a sequenceof nucleic acids into a host microorganism or cell. Only when thesequence of nucleic acids becomes stably replicated by the cell does thehost microorganism or cell become “transformed.” As will be appreciatedby those of ordinary skill in the art, “transformation” may take placeeither by incorporation of the sequence of nucleic acids into thecellular genome, i.e., chromosomal integration, or by extrachromosomalintegration. In contrast, an expression vector, e.g., a virus, is“infective” when it transduces a host microorganism, replicates, and(without the benefit of any complementary virus or vector) spreadsprogeny expression vectors, e.g., viruses, of the same type as theoriginal transducing expression vector to other microorganisms, whereinthe progeny expression vectors possess the same ability to reproduce.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free of components that normally accompanyit in its native state.

As used herein, the terms “nucleic acid sequence,” “sequence of nucleicacids,” and variations thereof shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones, provided thatthe polymers contain nucleobases in a configuration that allows for basepairing and base stacking, as found in DNA and RNA. Thus, these termsinclude known types of nucleic acid sequence modifications, for example,substitution of one or more of the naturally occurring nucleotides withan analog; internucleotide modifications, such as, for example, thosewith uncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), with negatively charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), and withpositively charged linkages (e.g., aminoalklyphosphoramidates,aminoalkylphosphotriesters); those containing pendant moieties, such as,for example, proteins (including nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.); those with intercalators (e.g.,acridine, psoralen, etc.); and those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.). As used herein, thesymbols for nucleotides and polynucleotides are those recommended by theIUPAC-IUB Commission of Biochemical Nomenclature (Biochemistry 9:4022,1970).

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter) and asecond nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

“Polyketides” refers to a large group of natural products that arederived from successive condensations of simple carboxylates, such asacetate, propionate or butyrate. Naturally occurring polyketides possessa broad range of biological activities, including antibiotics such astetracyclines and erythromycin, anticancer agents such as daunomycin andbryostatin, immunosuppressants such as FK506 and rapamycin, andveterinary products such as monensin and avermectin. Polyketides areproduced in most groups of organisms and are especially abundant in aclass of mycelial bacteria, the actinomycetes, which produce varioustypes of polyketides.

“Substrate for the polyketide synthase” refers to any substrate ontowhich a polyketide synthase is competent to condense an extender.Exemplary extenders include halomalonyl and haloacetyl moieties. Anexemplary halo moiety is fluoro.

The term “non-toxic” refers to an amount of a compound insufficient todestroy 50% of the cells of a population of a host microorganism. Thecompound may be present in an amount sufficient to inhibit or evenarrest cell growth, but allows polyketide formation to occurefficiently.

Also, and more generally, in accordance with disclosures, discussions,examples and embodiments herein, there may be employed conventionalmolecular biology, cellular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. (See, e.g., Sambrook, et al., “MolecularCloning: A Laboratory Manual,” Third Edition 2001 (volumes 1-3), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal CellCulture, R. I. Freshney, ed., 1986.)

III. The Compositions

In an exemplary embodiment, the invention provides a halogenatedpolyketide, or an analogue thereof. An exemplary halogenated polyketideis enzymatically synthesized either by a host microorganism or by one ormore isolated enzymes by condensation of a substrate for a polyketidesynthase and an extender according to Formula I:

in which X is selected from F, Cl, Br and I.

In various embodiments, the extender according to Formula I is preparedfrom a precursor according to Formula II:

in which X is as described above.

In an exemplary embodiment, the precursor according to Formula II isconverted to the compound according to Formula I by the action ofmalonyl-CoA sythetase (MatB). The conversion can occur using isolatedenzymes or within a host microorganism (e.g., an engineered hostmicroorganism) expressing one or more of these enzymes. In an exemplaryembodiment, MatB is a wild type enzyme from E. coli. In anotherembodiment, MatB is a non-naturally occurring enzyme.

In another embodiment, the compound according to Formula I is producedby enzymatic conversion of a precursor acetyl compound of Formula III tothe corresponding malonyl compound:

in which X is as described above.

In an exemplary embodiment, the compound according to Formula III isconverted to the compound according to Formula I by the action ofacetate kinase, phosphotransacetylase and the AccABCD subunits that makeup acetyl coenzyme A carboxylase (ACCase). The conversion can occurusing isolated enzymes or within a host microorganism (e.g., anengineered host microorganism) expressing one or more of these enzymes.In an exemplary embodiment, the AckA, Pta and/or the ACCase is a wildtype enzyme from E. coli. In another embodiment, the AckA, Pta and/orACCase is a non-naturally occurring enzyme.

In another embodiment, the extender according to Formula I is producedby conversion of the percussor of Formula III by the action of acetatekinase (AckA) and phosphotransacetylase (Pta). The conversion can occurusing isolated enzymes or within a host microorganism (e.g., anengineered host microorganism) expressing this enzyme. In an exemplaryembodiment, one or both of the AckA and Pta is (are) a wild type enzymefrom E. coli. In another embodiment, one or both of the AckA and Pta is(are) a non-naturally occurring enzyme.

In various embodiments, the precursor of Formula III is converted to theextender of Formula I using acyl-CoA synthase. The conversion can occurusing isolated enzymes or within a host microorganism (e.g., anengineered host microorganism) expressing this enzyme. In an exemplaryembodiment, the acyl-CoA synthase is a wild type enzyme from E. coli. Inanother embodiment, the acyl-CoA synthase is a non-naturally occurringenzyme.

In an exemplary embodiment, the invention provides a polyketide synthasefunctionalized with a halomalonyl moiety. In various embodiments, thehalomalonyl moiety is derived from a compound of Formula I and istransferred to the polyketide synthase via the action of a trans-acyltransferase.

In an exemplary embodiment, the invention provides a host microorganismcell that includes any one or more of the above-described components inan internal space of the host microorganism cell.

Polyketide Synthase

The enzymes responsible for the biosynthesis of polyketides are calledpolyketide synthases (PKSs). Two general classes of PKSs exist. Oneclass, known as Type I PKSs, is represented by the PKSs for thesynthesis of macrolide polyketides such as erythromycin and rapamycin.This type of PKSs has a modular enzymatic structure, in which a moduleis defined as a set of enzymatic domains that are necessary to catalyzethe recognition and incorporation of a specific 2-carbon extending unit(usually a malonyl-CoA, a methyl malonyl-CoA or a propionyl-CoA) intothe growing polyketide chain. A minimal type I PKS module contains threeenzymatic domains: (1) a ketosynthase domain (KS) which is responsiblefor catalyzing the Claisen condensation reaction between a starter unitor a growing polyketide chain and an extender unit; (2) anacyltransferase domain (AT) which selectively binds a specific extenderunit from the intracellular pools of the various CoA carboxylates andthen transfers it to the acyl carrier center; (3) an acyl carrierprotein domain (ACP) which contains a serine residue that has beenpost-translationally modified with a 4-phosphopantethein group andserves as the acceptor for the extender unit or a growing polyketidechain. In addition to the KS, AT, and ACP domains, a type I PKS modulecan also have one, two or three of the following domains: aketoreductase domain (KR) which reduces the β-ketone to the hydroxylfunction, a dehydratase domain (DH) which eliminates water from the α,βcarbon centers to generate a double bond between them, and aenoylreductase domain (ER) which further reduces the double bondgenerated by DH domain to yield the β-methylene group.

A co-linear relationship exists between the primary organization of theType I PKS and the structure of the polyketide backbone. For examples,the number of modules in the PKS determines the number of the two-carbonunits in the carbon backbone of the final polyketide product, thepresence of a specific AT domain determines which extender (malonate,methylmalonate or ethylmalonate, etc.) is incorporated into the growingpolyketide chain, and the presence of the reduction domains (KR, DH andER) in a module determines the extent of reduction of the .beta.-carbonformed at the give condensation.

The second class of PKSs, called Type II PKSs, is responsible for thesynthesis of aromatic polyketides such as daunorubicin andtetracenomycin. Type II PKSs have a single set of enzymatic activities(KS, AT, ACP, KR, etc.) that reside in individual proteins and are usediteratively to generate polyketides with polycyclic ring structure.There is no clear correlation between the type II PKS enzymaticorganization and the final polyketide structure.

The present invention provides a method of using the polyketide synthase(PKS) pathway to synthesize halogenated natural product scaffolds. ThePKS of use in the invention may be naturally occurring or non-naturallyoccurring. In some embodiments, the PKS is a hybrid PKS comprising of acombination of naturally occurring modules which in nature are not foundin this combination. In various embodiments, the PKS is naturallyoccurring but is heterologous to the host microorganism. In an exemplaryembodiment, the PKS is encoded by heterologous DNA.

Chain length is determined by the number of condensations that takeplace, which in turn is determined by the number of modules employed.All chain growth uses a starter determined by the loading module,typically contributing two (S1) or three (S2) carbon atoms to theoverall length of the acyl chain. Each extender module (e.g., thecompound of Formula I) contributes two carbons to the backbone and ahalogen. In various embodiments, the extender unit is halogenated andcontributes carbons in addition to those contributed by the compound ofFormula I (e.g., the extender molecule is a higher order homologue of acompound of Formula I).

In various embodiments, the invention provides a method in which asingle condensation yields a molecule with 4 carbon atoms (modules S1and D); two condensations generate a molecule that contains 6 carbons.Similarly, three condensations will yield molecules with 8 carbonbackbones.

In an exemplary embodiment, the invention utilizes a compound accordingto Formula I in at least one cycle of chain extension and ketoreduction,at least two cycles, at least three cycles, or at least four cycles. Inan exemplary embodiment, the at least one cycle generates a compoundwith a 2-halo-3-keto motif.

Any polyketide synthase, whether naturally occurring or non-naturallyoccurring can be used in practicing the methods of the invention. In anexemplary embodiment, the polyketide synthase is 6-deoxyethrythrolinideB synthase (DEBS)

The PKS can reside within a host cell, or it can be isolated orpurified. The PKS can synthesize the compound having an extended productwith a 2-halo-3-keto motif in vivo (in a host microorganism) or in vitro(in a cell extract or where all necessary chemical components orstarting materials are provided). The present invention provides methodsof producing the extended product using any of these in vivo or in vitromeans.

The level of reduction is also determined by the modules employed. Ingeneral, if more reduced molecules are desired, modules D, J, or onefrom the H group should be used. If a hydroxyl is desired internally toenable the formation of a lactone, a module from the B or F group shouldbe used. Lactone formation will occur if a PKS thioesterase domain (e.g.eryTE) is placed immediately downstream of the terminal external module.

Engineered Enzymes and Host Cells

The present invention provides for incorporation into a hostmicroorganism of a recombinant nucleic acid encoding an enzyme of use incarrying out the process of the invention. As will be apparent to thoseof skill in the art, exemplary enzymes include acetate kinase (AckA),phosphotransacetylase (Pta), one or more subunit (e.g., AccABCD) ofacetyl-CoA carboxylase, malonyl-CoA synthetase and polyketide synthase(PKS). This aspect of the invention is illustrated in a non-limitingmanner by reference to PKS.

Analogues of a naturally occurring PKS are prepared by manipulation ofthe relevant genes. A large number of modular PKS gene clusters havebeen mapped and/or sequenced, including erythromycin, soraphen A,rifamycin, and rapamycin, which have been completely mapped andsequenced, and FK506 and oleandomycin which have been partiallysequenced, and candicidin, avermectin, and nemadectin which have beenmapped and partially sequenced. Additional modular PKS gene clusters areexpected to be available as time progresses. These genes can bemanipulated using standard techniques to delete or inactivate activityencoding regions, insert regions of genes encoding correspondingactivities from the same or different PKS system, or otherwise mutatedusing standard procedures for obtaining genetic alterations.

Mutations can be made to the native sequences using conventionaltechniques. The substrates for mutation can be an entire cluster ofgenes or only one or two of them; the substrate for mutation may also beportions of one or more of these genes. Techniques for mutation includepreparing synthetic oligonucleotides including the mutations andinserting the mutated sequence into the gene encoding a PKS subunitusing restriction endonuclease digestion. (See, e.g., Kunkel, T. A. ProcNatl Acad Sci USA (1985) 82:448; Geisselsoder et al. BioTechniques(1987) 5:786.) Alternatively, the mutations can be effected using amismatched primer (generally 10-20 nucleotides in length) whichhybridizes to the native nucleotide sequence (generally cDNAcorresponding to the RNA sequence), at a temperature below the meltingtemperature of the mismatched duplex. The primer can be made specific bykeeping primer length and base composition within relatively narrowlimits and by keeping the mutant base centrally located. Zoller andSmith, Methods Enzymol. (1983) 100:468. Primer extension is effectedusing DNA polymerase, the product cloned and clones containing themutated DNA, derived by segregation of the primer extended strand,selected. Selection can be accomplished using the mutant primer as ahybridization probe. The technique is also applicable for generatingmultiple point mutations. See, e.g., Dalbie-McFarland et al. Proc NatlAcad Sci USA (1982) 79:6409. PCR mutagenesis will also find use foreffecting the desired mutations.

If replacement of a particular target region in a host polyketidesynthase is to be made, this replacement can be conducted in vitro usingsuitable restriction enzymes or can be effected in vivo usingrecombinant techniques involving homologous sequences framing thereplacement gene in a donor plasmid and a receptor region in a recipientplasmid. Such systems, advantageously involving plasmids of differingtemperature sensitivities are described, for example, in PCT publicationWO 96/40968.

The vectors used to perform the various operations to replace theenzymatic activity in the host PKS genes or to support mutations inthese regions of the host PKS genes may be chosen to contain controlsequences operably linked to the resulting coding sequences in a mannerthat expression of the coding sequences may be effected in a appropriatehost. However, simple cloning vectors may be used as well.

The recombinant nucleic acid can be a double-stranded or single-strandedDNA, or RNA. The recombinant nucleic acid can encode an open readingframe (ORF) of the PKS of the present invention. The recombinant nucleicacid can also comprise promoter sequences for transcribing the ORF in asuitable host cell. The recombinant nucleic acid can also comprisesequences sufficient for having the recombinant nucleic acid stablyreplicate in a host cell. The recombinant nucleic acid can be repliconcapable of stable maintenance in a host cell. In some embodiments, thereplicon is a plasmid. The present invention also provides for a vectoror expression vector comprising a recombinant nucleic acid of thepresent invention.

It will be apparent to one of skill in the art that a variety ofrecombinant vectors can be utilized in the practice of aspects of theinvention. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosethat integrate into the host cell genome.

The vectors may be chosen to contain control sequences operably linkedto the resulting coding sequences in a manner that expression of thecoding sequences may be effected in an appropriate host. Suitablecontrol sequences include those that function in eukaryotic andprokaryotic host cells. If the cloning vectors employed to obtain PKSgenes encoding derived PKS lack control sequences for expressionoperably linked to the encoding nucleotide sequences, the nucleotidesequences are inserted into appropriate expression vectors. This can bedone individually, or using a pool of isolated encoding nucleotidesequences, which can be inserted into host vectors, the resultingvectors transformed or transfected into host cells, and the resultingcells plated out into individual colonies. Suitable control sequencesfor single cell cultures of various types of organisms are well known inthe art. Control systems for expression in yeast are widely availableand are routinely used. Control elements include promoters, optionallycontaining operator sequences, and other elements depending on thenature of the host, such as ribosome binding sites. Particularly usefulpromoters for prokaryotic hosts include those from PKS gene clustersthat result in the production of polyketides as secondary metabolites,including those from Type I or aromatic (Type II) PKS gene clusters.Examples are act promoters, tcm promoters, spiramycin promoters, and thelike. However, other bacterial promoters, such as those derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) andmaltose, are also useful. Additional examples include promoters derivedfrom biosynthetic enzymes such as for tryptophan (trp), the β-lactamase(bla), bacteriophage lambda PL, and T5. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can beused.

As noted, particularly useful control sequences are those whichthemselves, or with suitable regulatory systems, activate expressionduring transition from growth to stationary phase in the vegetativemycelium. Illustrative control sequences, vectors, and host cells ofthese types include the modified S. coelicolor CH999 and vectorsdescribed in PCT Publication No. WO 96/40968 and similar strains of S.lividans. See U.S. Pat. Nos. 5,672,491; 5,830,750; 5,843,718; and6,177,262, each of which is hereby incorporated by reference. Otherregulatory sequences may also be desirable which allow for regulation ofexpression of the PKS sequences relative to the growth of the host cell.Regulatory sequences are known to those of skill in the art, andexamples include those which cause the expression of a gene to be turnedon or off in response to a chemical or physical stimulus, including thepresence of a regulatory compound. Other types of regulatory elementsmay also be present in the vector, for example, enhancer sequences.

Selectable markers can also be included in the recombinant expressionvectors. A variety of markers are known which are useful in selectingfor transformed cell lines and generally comprise a gene whoseexpression confers a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium. Such markersinclude, for example, genes that confer antibiotic resistance orsensitivity to the plasmid.

In various embodiments polypeptides obtained by the expression of thepolynucleotide molecules of the present invention may have at leastapproximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identity to one or more amino acid sequences encoded by the genes and/ornucleic acid sequences described herein for the polyketidetolerance-related and biosynthesis pathways.

As a practical matter, whether any particular polypeptide is at least50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identicalto any reference amino acid sequence of any polypeptide described herein(which may correspond with a particular nucleic acid sequence describedherein), such particular polypeptide sequence can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set such that the percentage of identity iscalculated over the full length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

For example, in a specific embodiment the identity between a referencesequence (query sequence, i.e., a sequence of the present invention) anda subject sequence, also referred to as a global sequence alignment, maybe determined using the FASTDB computer program based on the algorithmof Brutlag, et al., (Comp. App. Biosci. 6:237-245 (1990)). Preferredparameters for a particular embodiment in which identity is narrowlyconstrued, used in a FASTDB amino acid alignment, are: ScoringScheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that arelateral to the N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. A determination of whether aresidue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence are considered for thismanual correction. For example, a 90 amino acid residue subject sequenceis aligned with a 100 residue query sequence to determine percentidentity. The deletion occurs at the N-terminus of the subject sequenceand therefore, the FASTDB alignment does not show a matching/alignmentof the first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for.

For various embodiments of the invention the genetic manipulations maybe described to include various genetic manipulations, including thosedirected to change regulation of, and therefore ultimate activity of, anenzyme or enzymatic activity of an enzyme identified in any of therespective pathways. Such genetic modifications may be directed totranscriptional, translational, and post-translational modificationsthat result in a change of enzyme activity and/or selectivity underselected and/or identified culture conditions and/or to provision ofadditional nucleic acid sequences such as to increase copy number and/ormutants of an enzyme related to polyketide production. Specificmethodologies and approaches to achieve such genetic modification arewell known to one skilled in the art, and include, but are not limitedto: increasing expression of an endogenous genetic element; decreasingfunctionality of a repressor gene; introducing a heterologous geneticelement; increasing copy number of a nucleic acid sequence encoding apolypeptide catalyzing an enzymatic conversion step to produce apolyketide; mutating a genetic element to provide a mutated protein toincrease specific enzymatic activity; over-expressing; under-expressing;over-expressing a chaperone; knocking out a protease; altering ormodifying feedback inhibition; providing an enzyme variant comprisingone or more of an impaired binding site for a repressor and/orcompetitive inhibitor; knocking out a repressor gene; evolution,selection and/or other approaches to improve mRNA stability as well asuse of plasmids having an effective copy number and promoters to achievean effective level of improvement. Random mutagenesis may be practicedto provide genetic modifications that may fall into any of these orother stated approaches. The genetic modifications further broadly fallinto additions (including insertions), deletions (such as by a mutation)and substitutions of one or more nucleic acids in a nucleic acid ofinterest. In various embodiments a genetic modification results inimproved enzymatic specific activity and/or turnover number of anenzyme. Without being limited, changes may be measured by one or more ofthe following: K_(M); K_(cat); and K_(avidity).

In various embodiments, to function more efficiently, a microorganismmay comprise one or more gene deletions. Such gene disruptions,including deletions, are not meant to be limiting, and may beimplemented in various combinations in various embodiments. Genedeletions may be accomplished by mutational gene deletion approaches,and/or starting with a mutant strain having reduced or no expression ofone or more of these enzymes, and/or other methods known to thoseskilled in the art. Gene deletions may be effectuated by any of a numberof known specific methodologies, including but not limited to the RED/ETmethods using kits and other reagents sold by Gene Bridges (Gene BridgesGmbH, Dresden, Germany, <<www.genebridges.com>>).

Targeted deletion of parts of microbial chromosomal DNA or the additionof foreign genetic material to microbial chromosomes may be practiced toalter a host cell's metabolism so as to reduce or eliminate productionof undesired metabolic products. This may be used in combination withother genetic modifications such as described herein in this generalexample. In this detailed description, reference has been made tomultiple embodiments and to the accompanying drawings in which is shownby way of illustration specific exemplary embodiments in which theinvention may be practiced. These embodiments are described insufficient detail to enable those skilled in the art to practice theinvention, and it is to be understood that modifications to the variousdisclosed embodiments may be made by a skilled artisan.

Further, for polyketide production, such genetic modifications may bechosen and/or selected for to achieve a higher flux rate through certainenzymatic conversion steps within the respective polyketide productionpathway and so may affect general cellular metabolism in fundamentaland/or major ways.

It will be appreciated that amino acid “homology” includes conservativesubstitutions, i.e. those that substitute a given amino acid in apolypeptide by another amino acid of similar characteristics. Typicallyseen as conservative substitutions are the following replacements:replacements of an aliphatic amino acid such as Ala, Val, Leu and Ilewith another aliphatic amino acid; replacement of a Ser with a Thr orvice versa; replacement of an acidic residue such as Asp or Glu withanother acidic residue; replacement of a residue bearing an amide group,such as Asn or Gln, with another residue bearing an amide group;exchange of a basic residue such as Lys or Arg with another basicresidue; and replacement of an aromatic residue such as Phe or Tyr withanother aromatic residue.

For all nucleic acid and amino acid sequences provided herein, it isappreciated that conservatively modified variants of these sequences areincluded, and are within the scope of the invention in its variousembodiments. Functionally equivalent nucleic acid and amino acidsequences (functional variants), which may include conservativelymodified variants as well as more extensively varied sequences, whichare well within the skill of the person of ordinary skill in the art,and microorganisms comprising these, also are within the scope ofvarious embodiments of the invention, as are methods and systemscomprising such sequences and/or microorganisms. In various embodiments,nucleic acid sequences encoding sufficiently homologous proteins orportions thereof are within the scope of the invention. More generally,nucleic acids sequences that encode a particular amino acid sequenceemployed in the invention may vary due to the degeneracy of the geneticcode, and nonetheless fall within the scope of the invention.

As indicated herein, polypeptides having a variant amino acid sequencecan retain enzymatic activity. Such polypeptides can be produced bymanipulating the nucleotide sequence encoding a polypeptide usingstandard procedures such as site-directed mutagenesis or various PCRntechniques. As noted herein, one type of modification includes thesubstitution of one or more amino acid residues for amino acid residueshaving a similar chemical and/or biochemical property. For example, apolypeptide can have an amino acid sequence set forth in an amino acidsequence listed or otherwise disclosed herein comprising one or moreconservative substitutions.

More substantial changes can be obtained by selecting substitutions thatare less conservative, and/or in areas of the sequence that may be morecritical, for example selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation; (b) the charge or hydrophobicity of thepolypeptide at the target site; or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce the greatestchanges in polypeptide function are those in which: (a) a hydrophilicresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine oralanine; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysine, arginine, or histidine, is substituted for (or by) anelectronegative residue, e.g., glutamic acid or aspartic acid; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions (or other deletions or additions) can beassessed for polypeptides having enzymatic activity by analyzing theability of the polypeptide to catalyze the conversion of the samesubstrate as the related native polypeptide to the same product as therelated native polypeptide. Accordingly, polypeptides having 5, 10, 20,30, 40, 50 or less conservative substitutions are provided by theinvention.

Compositions of the present invention, such as genetically modifiedmicroorganisms, comprise a production pathway for a chemical product inwhich malonyl-CoA is a substrate, and may also comprise one or moregenetic modifications to reduce the activity of enzymes encoded by oneor more of the fatty acid synthetase system genes to prevent shunting ofthis extender into the fatty acid synthesis pathway. The compositionsmay be used in the methods and systems of the present invention.

The various PKS nucleic acid sequences or nucleotide sequences, or amixture of such sequences, can be cloned into one or more recombinantvectors as individual cassettes, with separate control elements or underthe control of a single promoter. The PKS subunits or components caninclude flanking restriction sites to allow for the easy deletion andinsertion of other PKS subunits. The design of such restriction sites isknown to those of skill in the art and can be accomplished using thetechniques described above, such as site-directed mutagenesis and PCR.Methods for introducing the recombinant vectors of the present inventioninto suitable hosts are known to those of skill in the art and typicallyinclude the use of CaCl₂ or other agents, such as divalent cations,lipofection, DMSO, protoplast transformation, conjugation, andelectroporation.

Of the more than thirty PKSs examined, the correspondence between use ofmodules in the biosynthesis and the structure of the polyketide producedis fully understood both at the level of the protein sequence of the PKSand the DNA sequence of the corresponding genes. The programming ofmodules into polyketide structure can be identified by sequencedetermination. It is possible to clone (or synthesize) DNA sequencescorresponding to desired modules and transfer them as fully functioningunits to heterologous, otherwise non-polyketide producing hosts such asE. coli (Pfeifer, et al., Science 291:1790 (2001)) and Streptomyces(Kao, et al., Science 265:509 (1994)). Additional genes employed forpolyketide biosynthesis have also been identified. Genes that determinephosphopantetheine:protein transferase (PPTase) that transfer the4-phosphopantetheine cofactor of the ACP domains, commonly present inpolyketide producing hosts, have been cloned in E. coli and other hosts(Weissman, et al., Chembiochem 5:116 (2004)). Moreover, genes for theproduction of precursors such as methylmalonyl CoA and ethylmalonyl CoAhave also been identified and cloned in heterologous hosts. It is alsopossible to re-program polyketide biosynthesis to produce a compound ofdesired structure by either genetic manipulation of a single PKS or byconstruction of a hybrid PKS composed of modules from two or moresources (Weissman, et al., Chembiochem 5:116 (2004)).

Recombinant methods for manipulating modular PKS genes to make the PKSsof the present invention are described in U.S. Pat. Nos. 5,672,491;5,843,718; 5,830,750; 5,712,146; and 6,303,342; and in PCT publicationNos. WO 98/49315 and WO 97/02358; hereby incorporated by reference. Anumber of genetic engineering strategies have been used with variousPKSs to demonstrate that the structures of polyketides can bemanipulated to produce novel polyketides (see the patent publicationsreferenced supra and Hutchinson, 1998, Curr Opin Microbiol. 1:319-329,and Baltz, 1998, Trends Microbiol. 6:76-83; hereby incorporated byreference). In some embodiment, the components of the hybrid PKS arearranged onto polypeptides having interpolypeptide linkers that directthe assembly of the polypeptides into the functional PKS protein, suchthat it is not required that the PKS have the same arrangement ofmodules in the polypeptides as observed in natural PKSs. Suitableinterpolypeptide linkers to join polypeptides and intrapolypeptidelinkers to join modules within a polypeptide are described in PCTpublication No. WO 00/47724, hereby incorporated by reference.

The vast number of polyketide pathways that have been elucidated providea host of different options to produce the halogenated compounds of thepresent invention. While the products can be vastly different in sizeand functionality, all employ virtually the same strategy forbiosynthesis. In an exemplary embodiment, the exact interfaces betweennon-cognate enzyme partners are determined on a case-by-case basis.ACP-linker-KS and ACP-linker-TE regions from the proteins of interestare aligned to examine the least disruptive fusion point for the hybridsynthase. Genetic constructions employ sequence and ligation independentcloning (SLIC) so as to eliminate the incorporation of genetic“scarring”.

A partial list of sources of PKS sequences that can be used in makingthe PKSs of the present invention, for illustration and not limitation,includes Ambruticin (U.S. Pat. No. 7,332,576); Avermectin (U.S. Pat. No.5,252,474; MacNeil, et al., 1993, Industrial Microorganisms: Basic andApplied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp.245-256; MacNeil, et al., 1992, Gene 115: 119-25); Candicidin (FR0008)(Hu, et al., 1994, Mol. Microbiol. 14:163-72); Epothilone (U.S. Pat. No.6,303,342); Erythromycin (WO 93/13663; U.S. Pat. No. 5,824,513; Donadio,et al., 1991, Science 252:675-79; Cortes, et al., 1990, Nature348:176-8); FK506 (Motamedi, et al., 1998, Eur. J. Biochem. 256:528-34;Motamedi, et al., 1997, Eur. J. Biochem. 244:74-80); FK520 or ascomycin(U.S. Pat. No. 6,503,737; see also Nielsen, et al., 1991, Biochem.30:5789-96); Jerangolid (U.S. Pat. No. 7,285,405); Leptomycin (U.S. Pat.No. 7,288,396); Lovastatin (U.S. Pat. No. 5,744,350); Nemadectin(MacNeil, et al., 1993, supra); Niddamycin (Kakavas, et al., 1997, J.Bacteriol. 179:7515-22); Oleandomycin (Swan, et al., 1994, Mol. Gen.Genet. 242:358-62; U.S. Pat. No. 6,388,099; Olano, et al., 1998, Mol.Gen. Genet. 259:299-308); Oligomycin (Omura, et al., J., 2001, Proc.Natl. Acad. Sci. USA 98:12215-12220); Pederin (PCT publication No. WO2003/044186); Pikromycin (Xue, et al., 2000, Gene 245:203-211);Pimaricin (PCT publication No. WO 2000/077222); Platenolide (EP Pat.App. 791,656); Rapamycin (Schwecke, et al., 1995, Proc. Natl. Acad. Sci.USA 92:7839-43); Aparicio, et al., 1996, Gene 169:9-16); Rifamycin(August et al., 1998, Chemistry & Biology, 5: 69-79); Soraphen (U.S.Pat. No. 5,716,849; Schupp, et al., 1995, J. Bacteriology 177: 3673-79);Spiramycin (U.S. Pat. No. 5,098,837); Tylosin (EP 0 791,655; Kuhstoss etal., 1996, Gene 183:231-36; U.S. Pat. No. 5,876,991). Additionalsuitable PKS coding sequences are readily available to one skilled inthe art, or remain to be discovered and characterized, but will beavailable to those of skill (e.g., by reference to GenBank). Each of thereferences cited is hereby specifically and individually incorporated byreference.

Products produced by the methods of the invention include complexpolyketides. Complex polyketides comprise a large class of naturalproducts that are synthesized in bacteria (mainly members actinomycetefamily; e.g. Streptomyces), fungi and plants. Polyketides form theaglycone component of a large number of clinically important drugs, suchas antibiotics (e.g., erythromycin, tylosin), antifungal agents (e.g.,nystatin), anticancer agents (e.g., epothilone), immunosuppressives(e.g., rapamycin), etc. Though these compounds do not resemble eachother either in their structure or their mode of action, they share acommon basis for their biosynthesis, which is carried out by a group ofenzymes designated polyketide synthases.

The assembly of a loading module and at least two extender modules canbe done in E. coli. Compounds requiring halogenated acetyl CoA orhalogenated malonyl CoA as precursors can be made in E. coli hosts. Themodules can also be cloned in vectors that can be introduced into avariety of Streptomyces hosts (e.g., Streptomyces coelicolor).

Compounds requiring propionate (methylmalonate) precursors can be madein a variety of Streptomyces hosts which have ample supplies of theseprecursors. Alternatively, E. coli can be fed with propionate and theenzyme methylmalonyl CoA mutase can be cloned in an E. coli hostengineered to incorporate vitB 12.

Compounds which require module J for their synthesis will contain anethyl side chain and will employ 2-ethylmalonyl CoA as a precursor.Ethylmalonate is produced from the isomerization of butyrate. The genesencoding the enzymes in this pathway to produce this precursor can becloned into in a suitable E. coli. Numerous streptomycetes exist thatproduce ethylmalonyl CoA, some of which are suitable for cloning andexpression of PKS genes (e.g., Streptomyces fradiae).

Host Microorganisms

Generally, a microorganism used for the present invention may beselected from bacteria, cyanobacteria, filamentous fungi and yeasts.

For some embodiments, microbial hosts initially selected should alsoutilize sugars including glucose at a high rate. Most microbes arecapable of utilizing carbohydrates. However, certain environmentalmicrobes cannot utilize carbohydrates to high efficiency, and thereforewould not be suitable hosts for such embodiments that are intended forglucose or other carbohydrates as the principal added carbon source.

As the genomes of various species become known, the present inventionmay be easily applied to an ever-increasing range of suitablemicroorganisms. Further, given the relatively low cost of geneticsequencing, the genetic sequence of a species of interest may readily bedetermined to make application of aspects of the present invention morereadily obtainable (based on the ease of application of geneticmodifications to an organism having a known genomic sequence). Suchmodifications are within the scope of the invention.

Based on the various criteria described herein, suitable microbial hostsfor the bio-production of polyketides include, but are not limited to,any gram negative organisms, more particularly a member of the familyEnterobacteriaceae, such as E. coli, or Oligotropha carboxidovorans, orPseudomononas sp.; any gram positive microorganism, for example Bacillussubtilis, Lactobaccilus sp. or Lactococcus sp.; a yeast, for exampleSaccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and othergroups or microbial species.

In some embodiments a recombinant microorganism is utilized.

Media and Culture Conditions

In addition to an appropriate carbon source, bio-production media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forpolyketide production, or other products made under the presentinvention.

Another aspect of the invention regards media and culture conditionsthat comprise genetically modified microorganisms of the invention andoptionally supplements.

Typically cells are grown at a temperature in the range of about 25° C.to about 40° C. in an appropriate medium, as well as up to 70° C. forthermophilic microorganisms. Suitable growth media in the presentinvention are common commercially prepared media such as Luria Bertani(LB) broth, M9 minimal media, Sabouraud Dextrose (SD) broth, Yeastmedium (YM) broth, (Ymin) yeast synthetic minimal media, and minimalmedia as described herein, such as M9 minimal media. Other defined orsynthetic growth media may also be used, and the appropriate medium forgrowth of the particular microorganism will be known by one skilled inthe art of microbiology or bio-production science. In variousembodiments a minimal media may be developed and used that does notcomprise, or that has a low level of addition of various components, forexample less than 10, 5, 2 or 1 g/L of a complex nitrogen sourceincluding but not limited to yeast extract, peptone, tryptone, soyflour, corn steep liquor, or casein. These minimal medias may also havelimited supplementation of vitamin mixtures including biotin, vitamin B12 and derivatives of vitamin B 12, thiamin, pantothenate and othervitamins. Minimal medias may also have limited simple inorganic nutrientsources containing less than 28, 17, or 2.5 mM phosphate, less than 25or 4 mM sulfate, and less than 130 or 50 mM total nitrogen.

Bio-production media, which is used in embodiments of the presentinvention with genetically modified microorganisms, must containsuitable carbon substrates for the intended metabolic pathways. Asdescribed hereinbefore, suitable carbon substrates include carbonmonoxide, carbon dioxide, and various monomeric and oligomeric sugars.

Suitable pH ranges for the bio-production of polyketides are between pH3.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for theinitial condition. However, the actual culture conditions for aparticular embodiment are not meant to be limited by these pH ranges.

Bio-production of polyketides may be performed under aerobic,microaerobic, or anaerobic conditions, with or without agitation.

The amount of polyketide or other product(s), produced in abio-production media generally can be determined using a number ofmethods known in the art, for example, high performance liquidchromatography (HPLC), gas chromatography (GC), GC/Mass Spectroscopy(MS), or spectrometry.

Bio-Production Reactors and Systems

Fermentation systems utilizing methods and/or compositions according tothe invention are also within the scope of the invention.

Any of the microorganisms as described and/or referred to herein may beintroduced into an industrial bio-production system where themicroorganisms convert a carbon source into a selected chemical product,such as a polyketide, in a commercially viable operation. Thebio-production system includes the introduction of such a microorganisminto a bioreactor vessel, with a carbon source substrate andbio-production media suitable for growing the recombinant microorganism,and maintaining the bio-production system within a suitable temperaturerange (and dissolved oxygen concentration range if the reaction isaerobic or microaerobic) for a suitable time to obtain a desiredconversion of a portion of the substrate molecules to a polyketide.Industrial bio-production systems and their operation are well-known tothose skilled in the arts of chemical engineering and bioprocessengineering.

Bio-productions may be performed under aerobic, microaerobic, oranaerobic conditions, with or without agitation. The operation ofcultures and populations of microorganisms to achieve aerobic,microaerobic and anaerobic conditions are known in the art, anddissolved oxygen levels of a liquid culture comprising a nutrient mediaand such microorganism populations may be monitored to maintain orconfirm a desired aerobic, microaerobic or anaerobic condition. Whensyngas is used as a feedstock, aerobic, microaerobic, or anaerobicconditions may be utilized. When sugars are used, anaerobic, aerobic ormicroaerobic conditions can be implemented in various embodiments.

Further to types of industrial bio-production, various embodiments ofthe present invention may employ a batch type of industrial bioreactor.A classical batch bioreactor system is considered “closed” meaning thatthe composition of the medium is established at the beginning of arespective bio-production event and not subject to artificialalterations and additions during the time period ending substantiallywith the end of the bio-production event. Thus, at the beginning of thebio-production event the medium is inoculated with the desired organismor organisms, and bio-production is permitted to occur without addinganything to the system. Typically, however, a “batch” type ofbio-production event is batch with respect to the addition of carbonsource and attempts are often made at controlling factors such as pH andoxygen concentration. In batch systems the metabolite and biomasscompositions of the system change constantly up to the time thebio-production event is stopped. Within batch cultures cells moderatethrough a static lag phase to a high growth log phase and finally to astationary phase where growth rate is diminished or halted. Ifuntreated, cells in the stationary phase will eventually die. Cells inlog phase generally are responsible for the bulk of production of adesired end product or intermediate.

A variation on the standard batch system is the fed-batch system.Fed-batch bio-production processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe nutrients, including the substrate, are added in increments as thebio-production progresses. Fed-batch systems are useful when cataboliterepression is apt to inhibit the metabolism of the cells and where it isdesirable to have limited amounts of substrate in the media. Measurementof the actual nutrient concentration in Fed-Batch systems may bemeasured directly, such as by sample analysis at different times, orestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and fed-batch approaches are common and well known in the art andexamples may be found in Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., Deshpande, Mukund V., Appl. Biochem. Biotechnol.,36:227, (1992), and Biochemical Engineering Fundamentals, 2nd Ed. J. E.Bailey and D. F. Ollis, McGraw Hill, New York, 1986, herein incorporatedby reference for general instruction on bio-production.

In various embodiments, the invention is directed to a system forbioproduction of a polyketide as described herein. An exemplary systemcomprises: a fermentation tank suitable for microorganism cell culture;a line for discharging contents from the fermentation tank to anextraction and/or separation vessel; an extraction and/or separationvessel suitable for removal of a polyketide from cell culture waste; aline for transferring polyketide to a dehydration vessel; and adehydration vessel suitable for conversion of wet polyketide to drypolyketide. In various embodiments, the system includes one or morepre-fermentation tanks, distillation columns, centrifuge vessels, backextraction columns, mixing vessels, or combinations thereof.

The following examples are offered to illustrate certain embodiments ofthe invention and are not to be construed as limiting the invention tothese embodiments.

EXAMPLES Example 1 Materials and Methods

Commercial materials. Luria-Bertani (LB) Broth Miller, LB Agar Miller,Terrific Broth (TB), yeast extract, malt extract, glycerol, andtriethylamine (TEA) were purchased from EMD Biosciences (Darmstadt,Germany). Carbenicillin (Cb), isopropyl-D-thiogalactopyranoside (IPTG),phenylmethanesulfonyl fluoride (PMSF), tris(hydroxymethyl)aminomethanehydrochloride (Tris-HCl), sodium chloride, dithiothreitol (DTT),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), magnesiumchloride hexahydrate, kanamycin (Km), acetonitrile,N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), dichloromethane,ethyl acetate and ethylene diamine tetraacetic acid disodium dihydrate(EDTA), were purchased from Fisher Scientific (Pittsburgh, Pa.). S odiumfluoroacetate, coenzyme A trilithium salt (CoA), acetyl-CoA,malonyl-CoA, methylmalonyl-CoA, diethylfluoromalonate, malonic acid,methylmalonic acid, tris(2-carboxyethyl)phosphine (TCEP) hydrochloride,lithium hexamethyldisilazide solution (LiHMDS), phosphoenolpyruvate(PEP), adenosine triphosphate sodium salt (ATP), nicotinamide adeninedinucleotide reduced form dipotassium salt (NADH), nicotinamide adeninedinucleotide phosphate reduced form (NADPH), myokinase, pyruvate kinase,lactate dehydrogenase, poly(ethyleneimine) solution (PEI),5-fluorouracil, β-mercaptoethanol, sodium phosphate dibasic hepthydrate,chlorotrifluoromethane and N,N,N′,N′-tetramethyl-ethane-1,2-diamine(TEMED) were purchased from Sigma-Aldrich (St. Louis, Mo.). Formic acidwas purchased from Acros Organics (Morris Plains, N.J.).Acrylamide/Bis-acrylamide (30%, 37.5:1), electrophoresis grade sodiumdodecyl sulfate (SDS), Bio-Rad protein assay dye reagent concentrate andammonium persulfate were purchased from Bio-Rad Laboratories (Hercules,Calif.). Restriction enzymes, T4 DNA ligase, Antarctic phosphatase,Phusion DNA polymerase, T5 exonuclease, and Taq DNA ligase werepurchased from New England Biolabs (Ipswich, Mass.). Deoxynucleotides(dNTPs) and Platinum Taq High-Fidelity polymerase (Pt Taq HF) werepurchased from Invitrogen (Carlsbad, Calif.). PageRuler™ Plus prestainedprotein ladder was purchased from Fermentas (Glen Burnie, Md.).Oligonucleotides were purchased from Integrated DNA Technologies(Coralville, Iowa), resuspended at a stock concentration of 100 μM in 10mM Tris-HCl, pH 8.5, and stored at either 4° C. for immediate use or−20° C. for longer term use. DNA purification kits and Ni-NTA agarosewere purchased from Qiagen (Valencia, Calif.). Complete EDTA-freeprotease inhibitor was purchased from Roche Applied Science (Penzberg,Germany). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), Amicon Ultra 3,000 MWCO and 30,000 MWCOcentrifugal concentrators, 5,000 MWCO regenerated celluloseultrafiltration membranes, and LiChroCART 250-4 Purospher RP-18e HPLCcolumn were purchased from EMD Millipore (Billerica, Mass.). Deuteriumoxide and chloroform-d were purchased from Cambridge IsotopeLaboratories (Andover, Mass.). ¹⁹F NMR spectra were collected at 25° C.on Bruker AVQ-400 or AV-600 spectrometers at the College of ChemistryNMR Facility at the University of California, Berkeley or on a BrukerBiospin 900 MHz spectrometer at the QB3 Central California 900 MHz NMRFacility or on a Bruker AV-600 spectrometer equipped with aQCI-CryoProbe at Novartis Institutes for Biomedical Research(Emeryville, Calif.). Spectra were referenced to CFCl₃ (0 ppm) or5-fluorouracil (D₂O: −168.33 ppm vs. CFCl₃). NMR assignments were madebased on COSY, ¹³C-¹H HSQC, ¹³C-¹H HMBC and ¹⁹F-¹H HMBC spectra whereappropriate. High-resolution mass spectral analyses were carried out atthe College of Chemistry Mass Spectrometry Facility.

Bacterial Strains.

E. coli DH10B-T1^(R) and BL21(de3)T1^(R) were used for DNA constructionand heterologous protein production, respectively, except for DEBSmodules, which were heterologously expressed in E. coli BAP1 (Pfeifer,B. A., et al., Science 291:1790-1792 (2001)).

Gene and plasmid construction. Standard molecular biology techniqueswere used to carry out plasmid construction. All PCR amplifications werecarried out with Phusion or Platinum Taq High Fidelity DNA polymerases.For amplification of GC-rich sequences from S. coelicolor, PCR reactionswere supplemented with DMSO (5%) using the standard buffer rather thanGC buffer with primer annealing temperatures 8-10° C. below the T_(m).All constructs were verified by sequencing (Quintara Biosciences;Berkeley, Calif.).

The synthetic gene encoding NphT7 was optimized for E. coli class IIcodon usage and synthesized using PCR assembly (Table S1). Gene2Oligowas used to convert the gene sequence into primer sets using defaultoptimization settings (Table S1) (Rouillard, J. M., et al., NucleicAcids Res. 32:W176-W180 (2004)). To assemble the synthetic gene, eachprimer was added at a final concentration of 1 nM to the first PCRreaction (50 μL) containing 1×Pt Taq HF buffer (20 mM Tris-HCl, 50 mMKCl, pH 8.4), MgSO₄ (1.5 mM), dNTPs (250 nM each), and Pt Taq HF (5 U).The following thermocycler program was used for the first assemblyreaction: 95° C. for 5 min; 95° C. for 30 s; 55° C. for 2 min; 72° C.for 10 s; 40 cycles of 95° C. for 15 s, 55° C. for 30 s, 72° C. for 20 splus 3 s/cycle; these cycles were followed by a final incubation at 72°C. for 5 min. The second assembly reaction (50 μL) contained 16 ΞL ofthe unpurified first PCR reaction with standard reagents for Pt Taq HF.The thermocycler program for the second PCR was: 95° C. for 30 s; 55° C.for 2 min; 72° C. for 10 s; 40 cycles of 95° C. for 15 s, 55° C. for 30s, 72° C. for 80 s; these cycles were followed by a final incubation at72° C. for 5 min. The second PCR reaction (16 μL) was transferred againinto fresh reagents and run using the same program. Following geneconstruction, the DNA smear at the appropriate size was gel purified andused as a template for the rescue PCR (50 μL) with Pt Taq HF and rescueprimers under standard conditions. The resulting rescue product wasinserted into pBAD33 and confirmed by sequencing, then amplified usingthe nphT7 F1/R1 primer set (Table S1) and inserted into the NdeI site ofpET-16b using the Gibson protocol (Gibson, D. G., et al., Nat. Methods,6:343-345 (2009)).

pET16b-His₁₀-AckA.EC and pET16b-His₁₀-Pta.EC were constructed byamplification from pRSFDuet-ackA.pta using the AckA.EC F/R and Pta.ECF/R primer sets (Table S1) and insertion into the NdeI-XhoI (Pta, GeneID 12872491) or NdeI-BamHI (AckA, Gene ID 12874027) sites of pET16b.pET28a-His₆-MatB.SCo and pET28a-His₆-Epi.SCo were constructed byamplification from S. coelicolor A3(2) M145 (ATCC BAA-471) genomic DNAusing the MatB.SCo F/R and epi.SCo F/R primer sets (Table S1) andinsertion into the NdeI-XhoI sites of pET28a. pET16x-His₁₀-THNS wasconstructed by amplification out of S. coelicolor genomic DNA using theTHNS F/R primer set (Table S1) and insertion into the NdeI-SpeI sites ofpET16x. pCDFDuet-DszsAT.SCe-MatB.SCo and pCDFDuet-ø-MatB.SCo wereconstructed by amplification from pET28a-His₆-MatB.SCo and pFW3 (Wong,F. T., et al., Biochemistry, 49:95-102 (2009)) using the pCDF-MatB.SCoF/R and pCDF-DszsAT.SCe F/R primer sets (Table S1) and insertion ofDszsAT.SCe and MatB.SCo into the NcoI-HindIII and NdeI-KpnI sites ofpCDFDuet-1 respectively. pTRC33-NphT7-PhaB was constructed by amplifyingNphT7 from pET-16b-NphT7 using the NphT7 G F/G R primer set (Table S1)and PhaB from pBT33-PhaABC (Bond-Watts, B. B., et al., Nat. Chem. Biol.,7:222-227 (2011)) using the PhaB F/R primer set (Table S1) where eachforward primer included the RBS from pET16b, then inserting both genessimultaneously into the BamHI-XbaI sites of pTRC33 using the Gibsonprotocol. pSV272-His₆-MBP-DEBS_(Mod2) andpSV272-His₆-MBP-DEBS_(Mod2)/AT⁰ (S2652A based on EryAI numbering) wereconstructed by amplification from pBP19 (Tsuji, S. Y., et al.,Biochemistry, 40:2326-2331 (2001)) using the MBP-M2 F/R primer set(pSV272-MBP-DEBS_(Mod2)-His₆) (Table S1) or MBP-M2 F/MBP-M2ATnull R andMBP-M2ATnull F/MBP-M2 R (pSV272-MBP-DEBS_(Mod2)/AT⁰-His₆) (Table S1) andinsertion into the SfoI-HindIII sites of pSV272.1 using the Gibsonprotocol. pBAD33.BirA.EC was cloned by the QB3 Macrolab.

Expression of his-Tagged Proteins.

TB (1 L) containing carbenicillin, kanamycin, and chloramphenicol (50μg/mL) as appropriate in a 2.8 L Fernbach baffled shake flask wasinoculated to OD₆₀₀=0.05 with an overnight TB culture of freshlytransformed E. coli containing the appropriate overexpression plasmid.The cultures were grown at 37° C. at 250 rpm to OD₆₀₀=0.6 to 0.8 atwhich point cultures were cooled on ice for 20 min, followed byinduction of protein expression with IPTG (His₁₀-AckA, His₁₀-Pta: 1 mM;His₁₀-AccA/B/C/D, His₁₀-THNS, DEBS_(Mod6)/AT⁰+TE-His₆[pAYC138, (Wong, F.T., et al., Biochemistry, 49:95-102 (2009))]: 0.4 mM; His₆-MatB,His₆-Epi, His₁₀-NphT7, DszsAT-His₆[pFW3, (Wong, F. T., et al.,Biochemistry, 49:95-102 (2009))], DEBS_(Mod6)+TE-His₆[pRSG54, (Wu, N.,et al., J. Am. Chem. Soc., 122:4847-4852 (2000))],DEBS_(Mod3)+TE-His₆[pRSG34, (Gokhale, R. S., et al., Science,284:482-485 (1999))], DEBS_(Mod3)/AT^(Q)FTE-His₆[pAYC136, (Wong, F. T.,et al., Biochemistry, 49:95-102 (2009))], MBP-DEBS_(Mod2)-His₆,MBP-DEBS_(Mod2)/AT⁰⁻-His₆: 0.2 mM) and overnight growth at 16° C. ForHis₁₀-AccB expression, pBAD33-BirA was co-expressed (L-arabinose, 0.2%)and the medium was supplemented with 20 nM D-(+)-biotin at induction.For MBP-DEBS_(Mod2)-His₆ and MBP-DEBS_(Mod2)/AT⁰-His₆, pRARE2 wasco-expressed. Cell pellets were harvested by centrifugation at 9,800×gfor 7 min at 4° C. and stored at −80° C.

Purification of His₁₀-AckA, His₁₀-Pta, His₆-MatB, His₆-Epi,His₁₀-AccA/B/C/D, DszsAT-His₆ and His₁₀-NphT7.

Frozen cell pellets were thawed and resuspended at 5 mL/g cell pastewith Buffer A (50 mM sodium phosphate, 300 mM sodium chloride, 20%glycerol, 20 mM BME, pH 7.5) containing imidazole (10 mM) forHis₁₀-AckA, His₁₀-Pta, His₁₀-AccA/B/C/D, His₁₀-NphT7, and DszsAT-His₆ orBuffer B (200 mM sodium phosphate, 200 mM sodium chloride, 30% glycerol,2.5 mM EDTA, 2.5 mM DTT, pH 7.5) for His₆-MatB and His₆-Epi. CompleteEDTA-free protease inhibitor cocktail (Roche) was added to the lysisbuffer before resuspension. The cell paste was homogenized before lysisby passage through a French Pressure cell (Thermo Scientific; Waltham,Mass.) at 14,000 psi. The lysate was centrifuged at 15,300×g for 20 minat 4° C. to separate the soluble and insoluble fractions. DNA wasprecipitated in the soluble fraction by addition of 0.15% (w/v)poly(ethyleneimine). The precipitated DNA was removed by centrifugationat 15,300×g for 20 min at 4° C. The remaining soluble lysate was dilutedthree-fold with Buffer A containing imidazole (10 mM) and loaded onto aNi-NTA agarose column (Qiagen, 1 mL resin/g cell paste) by gravity flowor on an AKTApurifier FPLC (2 mL/min; GE Healthcare; Piscataway, N.J.).The column was washed with Buffer A until the eluate reached anA_(280 nm)<0.05 or was negative for protein content by Bradford assay(Bio-Rad).

His₁₀-AckA, His₁₀-Pta, His₆-MatB, His₁₀-AccB, His₁₀-AccD, andDszsAT-His₆.

The column was washed with 5 to 10 column volumes with Buffer Asupplemented with imidazole (His₁₀-AckA, 40 mM; His₁₀-Pta, 35 mM;His₆-MatB, His₁₀-AccB, His₁₀-AccD, and DszsAT-His₆, 20 mM). The proteinwas then eluted with 300 mM imidazole in Buffer A.

His₆-Epi.

His₆-Epi was eluted using a linear gradient from 0 to 300 mM imidazolein Buffer A over 30 column volumes.

His₁₀-AccA, His₁₀-AccC and His₁₀-NphT7.

The column was washed with a linear gradient from 10 to 90 mM imidazolein Buffer A over 15 column volumes and then eluted with 300 mM imidazolein Buffer A.

Fractions containing the target protein were pooled by A_(280 nm) andconcentrated using either an Amicon Ultra spin concentrator (3 kDa MWCO,Millipore) or an Amicon ultrafiltration cell under nitrogen flow (65psi) using a membrane with an appropriate nominal molecular weightcutoff (Ultracel-5 or YM10, Millipore). Protein was then exchanged intoBuffer C (50 mM HEPES, 100 mM sodium chloride, 2.5 mM EDTA, 20%glycerol, pH 7.5) with (His₁₀-AckA, His₁₀-Pta, His₁₀-AccA/B/C/D,His₁₀-NphT7, DszsAT-His₆) or without (His₆-MatB and His₆-Epi) DTT (0.5-1mM) using a Sephadex G-25 column (Sigma-Aldrich, bead size 50-150 μm, 10mL resin/mL protein solution), then concentrated again before storage.

Final protein concentrations before storage were estimated using theA_(280 nm) calculated by ExPASY ProtParam as follows: His₁₀-AckA: 14.8mg/mL (A_(280 nm)=24,860 M⁻¹ cm⁻¹), His₁₀-Pta: 16.5 mg/mL(A_(280 nm)=37,360 M⁻¹ cm⁻¹), His₆-MatB: 19.8 mg/mL (A_(280 nm)=33,920M⁻¹ cm⁻¹), His₆-Epi: 18.5 mg/mL (A_(280 nm)=11,460 M⁻¹ cm⁻¹),His₁₀-AccA: 33.2 mg/mL (v_(280 nm)=25,900 M⁻¹ cm⁻¹), His₁₀-AccB: 23.0mg/mL (A_(280 nm)=2,980 M⁻¹ cm⁻¹), His₁₀-AccC: 32.6 mg/mL(A_(280 nm)=27,850 M⁻¹ cm⁻¹), His₁₀-AccD: 4.5 mg/mL (A_(280 nm)=16,960M⁻¹ cm⁻¹), DszsAT-His₆: 1.7 mg/mL (V_(280 nm)=17,420 M⁻¹ cm⁻¹),His₁₀-NphT7: 0.4 mg/mL (V_(280 nm)=26,930 M⁻¹ cm⁻¹). All proteins werealiquoted, flash-frozen in liquid nitrogen, and stored at −80° C.

Purification of DEBS_(Mod6)+TE-His₆, DEBS_(Mod6)/AT⁰+TE-His₆,DEBS_(Mod3)+TE-His₆, and DEBS_(Mod3)/AT⁰+TE-His₆.

The His-tagged DEBS module with thioesterase (DEBS_(Mod6)+TE) constructwas heterologously expressed in E. coli BAP1 pRSG54 as describedpreviously and purified using a modified literature protocol (Kumar, etal., Method. Enzymol., 269-293 (2004)). Cleared cell lysates wereprepared in Buffer B as described above, diluted three-fold with BufferA, and passed over a Ni-NTA agarose column (Qiagen, approximately 1 mL/gcell paste) on an ÄKTApurifier FPLC. The column was washed with Buffer Auntil the eluate reached an A_(280 nm)<0.05. Protein was eluted withBuffer D (50 mM sodium phosphate, 50 mM sodium chloride, 20 mM BME, 20%glycerol, 100 mM imidazole, pH 7.5). The eluate was diluted three-foldwith Buffer E (50 mM HEPES, 2.5 mM EDTA, 2.5 mM DTT, 20% glycerol, pH7.5), loaded onto a HiTrap Q HP column (GE Healthcare, 5 mL), and elutedwith a linear gradient from 0 to 1 M sodium chloride in Buffer E over 30column volumes (4.5 mL/min) Fractions containing the target protein(eluted at 350 mM sodium chloride) were pooled by A_(280 nm) andconcentrated under nitrogen flow (65 psi) in an Amicon ultrafiltrationcell using a YM10 membrane. The protein was flash-frozen in liquidnitrogen and stored at −80° C. at a final concentration of 6-30 mg/mL,which was estimated using the calculated A_(280 nm) (DEBS_(moo) andDEBS_(moo)/AT⁰: 203,280 M⁻¹ cm⁻¹; DEBS_(Mod6) and DEBS_(Mod6)/AT⁰:206,260 M⁻¹ cm⁻¹).

Purification of MBP-DEBS_(Mod2)-His₆ and MBP-DEBS_(Mod2)/AT⁰-His₆.

Cleared lysates were prepared as described for other DEBS modules,diluted three-fold with Buffer A containing 10 mM imidazole, and boundin batch to Ni-NTA resin (2.5 mL/g cell paste) for 2 h. The slurry waspoured into a fitted column and washed with Buffer A containing 10 mMimidazole until the eluate reached A_(280 nm)<0.05. The protein waseluted with Buffer A containing 300 mM imidazole and concentrated to 1mg/mL in an Amicon ultrafiltration cell using a YM10 (30 kD NMWL)membrane. The protein was then dialyzed overnight against Buffer Econtaining 50 mM NaCl with TEV protease (1 mg/100 mg protein substrate)to remove the MBP tag. The protein was loaded onto a HiTrap Q HP columnand eluted by a linear gradient from 0 to 500 mM NaCl in Buffer E over20 column volumes. Fractions containing the desired protein wereidentified by SDS-PAGE (eluting at 350 mM NaCl), pooled, andconcentrated in a YM10 (30 kD NMWL) Amicon Ultra spin concentrator.Protein aliquots were flash-frozen in liquid nitrogen and stored at −80°C. at a final concentration of 20-25 mg/mL, which was estimated usingthe calculated A_(280 nm) (158,360 M⁻¹ cm⁻¹).

Purification of His₁₀-THNS.

His₁₀-THNS was purified according to a modified literature procedure(Izumikawa, M., et al., J. Ind. Microbiol. Blot., 30:510-515 (2003)).Cleared cell lysates were prepared in Buffer F (50 mM Tris-HCl, 500 mMsodium chloride, 20 mM BME, 10% glycerol pH 8) supplemented withimidazole (10 mM), PMSF (0.75 mM), and Tween 20 (1% v/v) as describedabove, diluted with Buffer G containing imidazole (10 mM) and Tween 20(1%), and loaded onto a HisTrap FF column (GE Healthcare, 1 mL) on anAKTApurifier FPLC (1 mL/min) The column was washed with 10 mM followedby 20 mM imidazole in Buffer G, each time until the eluate reached anA_(280 nm)<0.05. Protein was eluted with 300 mM imidazole in Buffer Gand concentrated with an Amicon Ultra spin concentrator (10 kDa MWCO).His₁₀-THNS was then exchanged into Buffer C containing DTT (1 mM) usinga Sephadex G-25 column (Sigma-Aldrich, bead size 50-150 μm, 10 mL/mLprotein solution) and concentrated again. The protein was flash-frozenin liquid nitrogen and stored at −80° C. at a final concentration of 4.4mg/mL, which was estimated using the calculated A_(280 nm) (33,920 M⁻¹cm⁻¹).

ESI-MS screening method for acyl-CoAs. Preparative HPLC fractions werescreened on an Agilent 1290 HPLC system using a Zorbax Eclipse Plus C-18column (3.5 lam, 2.1×30 mm, Agilent) with a linear gradient from 0 to65% acetonitrile over 2 min with 0.1% formic acid as the aqueous mobilephase (0.75 mL/min) Mass spectra were recorded on an Agilent 6130 singlequadrupole MS with ESI source, operating in negative and positive ionscan mode.

Fluoromalonate.

Diethylfluoromalonate (0.5 mL, 3.2 mmol) was saponified with methanolicsodium hydroxide (2 M, 3.5 mL) in dichloromethane and methanol (9:1 v/v,32 mL) and the sodium salt isolated by filtration through a Büchnerfunnel with a fine porosity glass frit (Theodorou, V., et al.,Tetrahedron Lett., 48:8230-8233 (2007)). ¹⁹F NMR (565 MHz, D₂O,5-fluorouracil=−168.3 ppm): δ −176.43 (d, J=53 Hz).

Fluoromalonyl-CoA.

Fluoromalonyl-CoA was prepared enzymatically from fluoromalonate and CoAusing MatB and ATP. A myokinase/pyruvate kinase/PEP system was also usedto regenerate ATP in order to avoid high concentrations of AMP thatmight inhibit MatB. The reaction mixture (10 mL) contained 100 mM sodiumphosphate, pH 7.5, phosphoenolpyruvate (5 mM), TCEP (2.5 mM), magnesiumchloride (5 mM), fluoromalonate (2.5 mM), ATP (2.5 mM), pyruvate kinase(36 U), myokinase (20 U), CoA (2 mM) and MatB (5 μM). The mixture wasincubated at 37° C. for 6 h and then at room temperature for 16 h beforelyophilizing overnight. The residue was dissolved in water (1.6 mL) andacidified to pH ˜2 by addition of 70% perchloric acid (160 μL).Insoluble material was removed by centrifugation at 18,000×g for 10 min.The supernatant was adjusted to pH 6 by addition of 10 M sodiumhydroxide (100 μL) and desalted on an Agilent 1200 HPLC system using aZorbax Eclipse XDB C-18 column (5 μm, 9.4×250 mm, Agilent) with a lineargradient from 0 to 10% methanol over 9 min with 50 mM sodium phosphate,25 mM trifluoroacetic acid, pH 4.5 as the aqueous mobile phase (3mL/min) Fractions eluting near the void volume, containing bothfluoromalonyl-CoA and CoA, were lyophilized overnight, dissolved inwater (1 mL), and purified using a Zorbax Eclipse XDB C-18 column (5 μm,9.4×250 mm) with a linear gradient from 0 to 50% methanol over 45 mMwith 50 mM sodium phosphate, pH 4.5 as the aqueous mobile phase (3mL/min) Fractions were screened by ESI-MS and those containing purefluoromalonyl-CoA were lyophilized overnight, dissolved in water (1 mL),and desalted using a Zorbax Eclipse XDB C-18 column (5 μm, 9.4×250 mm)with a linear gradient from 0 to 15% acetonitrile over 30 min with wateras the mobile phase (3 mL/min) The desalted fluoromalonyl-CoA waslyophilized and redissolved in water or D₂O. The fluoromalonyl-CoAsolutions were stored at −20° C. but are stable for at least 24 h atroom temperature. During NMR measurements in D₂O, complete H-D exchangeoccurred at the fluorine-substituted carbon over the course of 48 h.Spectra are shown in FIG. 8. ¹H NMR (600 MHz, D₂O, MeOH=3.34 ppm): δ8.55 (s, 1H, H₈), 8.27 (s, 1H, H₂), 6.17 (d, J=6.6 Hz, 1H, H_(1′)), 5.23(d, J=50.3 Hz, 1H, O₂C—CHF—C═O), 4.89-4.79 (m, 2H, H_(2′) and H_(3′)),4.59 (m, 1H, H_(4′)), 4.23 (m, 2H, H_(5′)), 4.00 (s, 1H, H_(3″)), 3.82(dd, J=10.2, 4.6 Hz, 1H, pro-R—H_(1′)), 3.54 (dd, J=10.0, 4.4 Hz, 1H,pro-S— H_(1″)), 3.49-3.39 (m, 2H, H_(5″)), 3.38-3.29 (m, 2H, H_(8″)),3.11-3.02 (m, 2H, H_(9″)), 2.42 (t, J=6.7 Hz, 3H, H_(6″)), 0.88 (s, 3H,H_(10″)), 0.74 (s, 3H, H_(11′)). ¹³C NMR (226 MHz, D₂O, CH₃OH=49.15ppm): δ 196.92, 196.85 (d, J=27.5 Hz, CO₂), 175.02 (C_(4″)), 174.32(C_(7″)), 169.52 (d, J=21.0 Hz, O₂C-CDF-C═O), 155.37 (C₆), 152.40 (C₂),149.57 (C₄), 140.37 (C₈), 118.91 (C₅), 93.32 (td, J=25 Hz, 197 Hz,O₂C—CDF-C═O), 86.61 (C_(1′)), 83.81 (d, J=9 Hz, C_(4′)), 74.56 (d, J=5Hz, C_(3′) or C_(1″)), 74.35 (C_(3″)), 74.00 (d, J=5 Hz, C_(2′)), 72.16(d, J=6 Hz, C_(3′) or C_(1″)), 65.56 (C_(5′)), 38.66 (C_(5″) or C_(6″)),38.58 (d, J=8 Hz, C_(8″)) 35.58 (d, J=30 Hz, C_(9″)), 27.60 (d, J=3 Hz,C_(5″) or C_(6″)), 21.16 (C_(10″)), 18.22 (C_(11″)). ¹⁹F NMR (565 MHz,D₂O, CF₃CO₂H=−76.20 ppm): δ −182.11 (dd, J=7.8, 50.4 Hz, O₂C—CHF—C═O),−182.72 (m, O₂C—CDF—C═O). HR-ESI-MS [M−H]⁻: calculated forC₂₄H₃₆FN₂O₁₉P₃S, m/z, 870.0989. found m/z 870.0991.

Enzyme Assays.

Kinetic parameters (k_(cat), K_(M)) were determined by fitting the datausing Microcal Origin to the equation: v_(o)=v_(max) [S]/(K_(M)+[S]),where v is the initial rate and [S] is the substrate concentration. Dataare reported as mean±s.e. (n=3) unless otherwise noted with standarderror derived from the nonlinear curve fitting. Error bars on graphsrepresent mean±s.d. (n=3). Error in k_(cat)/K_(M) is calculated bypropagation of error from the individual kinetic parameters.

Acetyl-CoA Carboxylase.

ACCase activity was measured using a discontinuous HPLC assay. Assayswere performed at 30° C. in a total volume of 200 μL containing 50 mMHEPES, pH 7.5, TCEP (10 mM), bovine serum albumin (3 mg/mL), CoA (0.5mM), ATP (2.5 mM), magnesium chloride (10 mM), sodium bicarbonate (75mM), acetate or fluoroacetate (10 mM), phosphoenolpyruvate (10 mM),pyruvate kinase (4 U), AckA (0.1 μM), Pta (10 μM) and ACCase (15 μM).The pH of the buffer remained unchanged after addition of sodiumbicarbonate. ACCase stock solution was prepared by pre-mixing theprotein subunits at equimolar ratio (85 μM) except for AccB, which wasadded at 1.5-fold molar excess. The reaction was initiated with additionof ATP. Aliquots (20 μL) were removed and quenched by the addition of70% perchloric acid (1 μL). Insoluble material was removed bycentrifugation and the supernatant was analyzed on an Agilent 1200 or1290 HPLC system on a LiChroCART 250-4 Purospher RP-18e column (5 μm,4.6×250 mm, Millipore) and monitored at A_(260 nm). For reactions withacetate, a linear gradient from 2 to 20% acetonitrile over 10 min with 5mM sodium phosphate and 5 mM sodium citrate with 0.1% trifluoroaceticacid, pH 4.6 as the aqueous mobile phase (1 mL/min) was used to analyzethe reaction. For reactions with fluoroacetate, a linear gradient from 2to 15% acetonitrile containing 0.1% TEA over 15 min with 10 mM Tris, pH8.0 containing 0.1% TEA as the aqueous mobile phase (1 mL/min) was used.Buffers containing TEA were made fresh daily and could be used for atleast 6 h before significant change in chromatography was observed.

Malonyl-CoA Synthetase.

MatB activity was measured using a modified literature method(Williamson, J. R., et al., Method. Enzymol., 434-513 (1969)). Theproduction of AMP was coupled to pyruvate formation by myokinase andpyruvate kinase, which in turn was coupled to NADH oxidation by lactatedehydrogenase. Assays were performed at 30° C. in a total volume of 200μL containing 100 mM HEPES, pH 7.5, TCEP (1 mM), ATP (2.5 mM), magnesiumchloride (5 mM), phosphoenolpyruvate (1 mM), NADH (0.3 mM), myokinase(0.5 U), pyruvate kinase (3.6 U), lactate dehydrogenase (2.6 U),dicarboxylic acid (25 μM-1 mM malonate, 50 μM-10 mM fluoromalonate or 25μM-1.5 mM methylmalonate) and MatB (26 nM for malonate, 1 μM forfluoromalonate and 200 nM for methylmalonate). The reaction wasinitiated with addition of CoA (0.5 mM) and monitored at 340 nm in aBeckman Coulter DU-800 spectrophotometer.

Acetoacetyl-CoA Synthase.

NphT7 activity was measured using a NADPH-coupled assay with PhaB.Assays were performed at 30° C. in a total volume of 500 μL containing50 mM HEPES, pH 7.5, NADPH (160 μM), acetyl-CoA (200 μM), PhaB (0.05mg/mL), NphT7 (0.2 μM for malonyl-CoA; 0.5 μM for fluoromalonyl-CoA) andmalonyl-CoA (5-150 μM) or fluoromalonyl-CoA (5-200 μM). Reactions wereinitiated with the addition of malonyl- or fluoromalonyl-CoA andmonitored at 340 nm in an Agilent 8453 diode array spectrophotometer.The PhaB-coupled assay was tested both by doubling NphT7, which doubledthe initial velocity with both the fluorinated and non-fluorinatedsubstrates, and also by doubling the amount of PhaB, which led to nodifference in initial velocity.

Acyl-CoA Hydrolysis by DEBS.

Hydrolytic activity of DEBS_(Mod6)+TE was measured by monitoring thereaction of free CoA with DTNB as described previously (Huang, F., etal., Chem. Biol., 13:475-484 (2006)). Assays were performed at 37° C. ina total volume of 200 μL containing 400 mM sodium phosphate, pH 7.5, 500μM DTNB, and DEBS_(Mod6)+TE (1 μM). Reactions were initiated by additionof acyl-CoA (0.5 mM) and monitored at 412 nm in a Beckman Coulter DU-800spectrophotometer. Release of CoA was quantified by comparison to astandard curve (5-100 μM).

Tetrahydroxynaphthalene Synthase.

THNS activity was measured by monitoring THN productionspectrophotometrically at 340 nm as previously described (Izumikawa, M.,et al., J. Ind. Microbiol. Blot., 30:510-515 (2003)). The specificactivity was comparable to the reported value.

Tetrahydroxynaphthalene Production Using THNS

All reactions (160 μL) contained 100 mM HEPES, pH 7.5, magnesiumchloride (10 mM), BSA (300 μg/mL), sodium bicarbonate (75 mM),phosphoenolpyruvate (10 mM) and pyruvate kinase (2.9 U). The followingfinal concentrations of substrates and enzymes were added to theappropriate reactions: AckA (10 nM), Pta (10 μM), ACCase (5 μM), MatB (5μM), myokinase (1.6 U), sodium malonate (5 mM), malonyl-CoA (0.5 mM),acetyl-CoA (0.5 mM), and ATP (2.5 mM). ACCase was incubated at 30° C.for 10 min with the reaction mixture before initiation with THNS (2.5μM). When AckA/Pta activation of acetate was included, reaction mixtureswere pre-incubated at 30° C. for 6 min before addition of ACCase.Reactions containing TCEP (2.5 mM) were also tested, but no apparenteffect on THN production was observed. All reactions were incubated at30° C. for 24 h and flash-frozen in liquid nitrogen. Samples were thawedindividually on ice before quantifying total polyketide production usinga Beckman DU-800 spectrophotometer. A_(510 nm) was taken as a measure oftetrahydroxynaphthalene, flaviolin, and their spontaneous polymerizationproducts (Funa, N., et al., Nature, 400:897-899 (1999)).

2-fluoro-3-hydroxybutyryl-CoA production using NphT7

As acetofluoroacetyl-CoA proved to degrade fairly rapidly under theassay conditions, 2-fluoro-3-hydroxybutyryl-CoA was isolated from a 10mL reaction containing 100 mM HEPES, pH 7.5, fluoromalonate (10 mM), CoA(500 μM), NADPH (1 mM), ATP (1 mM), magnesium chloride (5 mM),phosphoenolpyruvate (10 mM), pyruvate kinase (180 U), myokinase (100 U),MatB (40 μM), PhaB (7 μM) and NphT7 (2 μM) that was initiated by theaddition of acetyl-CoA (0.5 mM, limiting reagent). The reaction wasincubated at 30° C. overnight followed by quenching by the addition of70% perchloric acid (50 μL). 2-fluoro-3-hydroxybutryl-CoA was purifiedusing a Zorbax Eclipse XDB C-8 column (5 μm, 9.4×250 mm, Agilent) with alinear gradient from 0 to 5% acetonitrile over 30 min (3 mL/min) with 50mM sodium phosphate with 0.1% trifluoroacetic acid (pH 4.5) as theaqueous mobile phase. Fractions containing 2-fluoro-3-hydroxybutryl-CoAwere identified by ESI-MS and lyophilized. The remaining solid wasdissolved in water (1 mL) and purified a second time a Zorbax EclipseXDB C-8 column (5 μm, 9.4×250 mm, Agilent) with a linear gradient from 0to 5% acetonitrile over 30 min (3 mL/min) with 0.1% formic acid as theaqueous mobile phase. Fractions containing 2-fluoro-3-hydroxybutryl-CoAwere identified by ESI-MS and lyophilized. Two diastereomers wereobserved by NMR in an approximately 2.5:1 ratio. ¹H NMR (600 MHz, D₂O,acetonitrile=2.06 ppm): δ 8.54 (s, 1H, H₈), 8.30 (s, 1H, H₂), 6.08 (d,J=5.9 Hz, 1H, H₁), 4.93 (dd, J=47.8, 2.6 Hz, 0.2H, HOCH—CHF—C═O minordiastereomer), 4.85 (dd, J=46.9, 2.2 Hz, 0.8H, HOCH—CHF—C═O majordiastereomer), 4.77-4.77 (m, 2H, H_(2′) and H_(3′)), 4.46 (m, 1H,H_(4′)), 4.16-4.04 (m, 3H, HOCH—CHF—C═O, H_(5′)), 3.89 (s, 1H, H_(3″)),3.72 (d, J=7.1 Hz, 1H, H_(1″) pro-R), 3.46 (d, J=9.7 Hz, 1H,pro-S—H_(1″) pro-S), 3.31 (t, J=6.5 Hz, 2H, H_(5″)), 3.26-3.20 (m, 2H,H_(8″)), 2.95 (m, 2H, H_(9″)), 2.30 (t, J=6.5 Hz, 2H, H_(6″)), 1.15 (d,J=6.7 Hz, 2H, H ₃C—HOCH—CHF major diastereomer), 1.05 (d, J=6.4 Hz, 1H,H ₃C—HOCH—CHF minor diastereomer), 0.79 (s, 3H, H_(10″)), 0.67 (s, 3H,H_(11″)). ¹⁹F NMR (565 MHz, D₂O, CF₃CO₂H=−76.20 ppm): δ −198.62 (dd,J=48.24, 23.3 Hz, HOCH—CHF—C═O), −206.85 (dd, J=46.9, 27.5,HOCH—CHF—C═O). ESI-MS [M+H]⁺: calculated for C₂₅H₄₂FN₇O₁₈P₃S, m/z,872.2. found m/z 872.0.

Triketide Lactone Production Using DEBS_(Mod6)+TE and MatB

Assay and preparative mixtures contained 400 mM sodium phosphate, pH7.5, phosphoenolpyruvate (50 mM), TCEP (5 mM), magnesium chloride (10mM), ATP (2.5 mM), pyruvate kinase (27 U/mL), myokinase (10 U/mL), CoA(0.5 mM), methylmalonyl-CoA epimerase (5 μM), MatB (40 μM or asspecified) and fluoro- or methylmalonate (10-20 mM). Including NADPHresulted in only trace yields of reduced triketide product, even withthe native methylmalonyl-CoA extender, so the cofactor was omitted. Thismixture was incubated at 37° C. for 30-45 min and then initiated byaddition of the N-acetylcysteamine thioester of(2S,3R)-2-methyl-3-hydroxypentanoic acid (NDK-SNAC, 1-10 mM) (Cane, D.E., et al., J. Am. Chem., 115:527-535 (1993)) and DEBS_(Mod6)+TE (10μM). Aliquots (35 μL) were removed and quenched by addition of 70%perchloric acid (1.75 μL). Samples were centrifuged at 18,000×g topellet the precipitated protein. The supernatant (33 μL) was removed andadded to 1 M sodium bicarbonate (6.6 μL) bringing the final pH to 4-5.Excess salts were precipitated by freezing in liquid nitrogen andcentrifuging at 18,000×g until thawed. The supernatant was removed andanalyzed on a Zorbax Eclipse XDB C-18 column (3.5 μm, 3×150 mm, 35° C.,Agilent) using a linear gradient from 0 to 40% acetonitrile over 14 minwith 0.1% formic acid as the aqueous mobile phase after an initial holdat 0% acetonitrile for 30 s (0.8 mL/min) Products were monitored usingan Agilent G1315D diode array detector (TKL, A_(260 nm) or A_(275 nm),F-TKL, A_(247 nm), NDK-SNAC; A_(260 nm)). The identity of each compoundwas verified using an Agilent 6130 single quadruple mass spectrometer innegative ion mode. For absolute quantification, each analyte wascompared to an external standard curve. The concentration of the2-fluoro-2-desmethyltriketide lactone (F-TKL) synthetic standard wasdetermined by ¹⁹F NMR using the ERETIC method (Akoka, S., et al., Anal.Chem., 71:2554-2557 (1999)) against an external standard of 5.00 mM5-fluorouracil. The triketide lactone (TKL) standard was preparedenzymatically, and the 2-desmethyltriketide lactone (H-TKL) standard wassynthesized as described (Hinterding, K., et al., Tetrahedron lett.,42:8463-8465 (2001)). The concentrations of TKL and H-TKL weredetermined by ¹H NMR in D₂O using the ERETIC method against an externalstandard of diethylfluoromalonate (75 mM).

Enzymatic Preparation of Methyl- and Fluorotriketide Lactones fromMethylmalonate and Fluoromalonate.

Reaction mixtures (TKL, 4 mL; F-TKL, 8 mL) containing NDK-SNAC (10 mM)were prepared as described above and incubated for 18 h. Protein wasremoved by the addition of 70% perchloric acid (0.05 volumes) andcentrifuged at 18,000×g for 10 min. The supernatant was removed andextracted extensively with dichloromethane (TKL, 5×15 mL; F-TKL, 5×30mL) and the organic layers concentrated to 5-10 mL by rotaryevaporation. The residue was transferred to a silanized glass vial(Sigmacote®, Sigma-Aldrich) and 50 mM sodium bicarbonate was added (1mL). The dichloromethane was removed from the biphasic mixture by rotaryevaporation to transfer the triketide into the aqueous phase. Theaqueous solution of triketide was purified on a Zorbax Eclipse XDB C-18column (5 μm, 9.4×250 mm, Agilent) using a linear gradient from 0 to27.5% methanol with 50 mM sodium phosphate, pH 4.5 over 45 min as theaqueous mobile phase (3 mL/min) Fractions containing triketide werepooled and extracted with dichloromethane (4×3 volumes), and thecombined organic layers were dried over magnesium sulfate andconcentrated.

The TKL was purified further on a Zorbax Eclipse XDB C-18 (5 μm, 9.4×250mm, Agilent) using a linear gradient from to 0 to 30% acetonitrile with0.1% formic acid as the aqueous mobile phase over 45 min (3 mL/min)after transferring back into bicarbonate buffer as described above.Fractions containing TKL were combined and lyophilized for analysis. Dueto the presence of a α-keto moiety, TKL was expected to be produced as adiastereomeric mixture, and was in fact isolated as a 100:7 mixture of(2R,4S,5R)-2,4-Dimethyl-3-oxo-5-hydroxy-n-heptanoic acid β-lactone andits 2S-epimer. The observed NMR spectra are in agreement with theliterature (Luo, G., et al., Bioorg. Med. Chem., 4:995-999 (1996)). ¹HNMR (500 MHz, CDCl₃): δ 4.66 (ddd, J=8.4, 5.4, 2.9 Hz, 2R H₅), 4.48-4.42(m, 2S H₅), 3.62 (q, J=6.6 Hz, 2R H₂), 3.25 (d, J=7.5 Hz, 2S H₂), 2.83(dd, J=7.2, 4.7 Hz, 2S H₄), 2.63 (qd, J=7.6, 2.9 Hz, 2R H₄), 1.93-1.82(m, 2R H_(6a)), 1.65 (dqd, J=14.8, 7.6, 5.4 Hz, 2R H_(6b)), 1.48 (d,J=7.5 Hz, 2S C₂—CH ₃), 1.37 (d, J=6.7 Hz, 2R C₂—CH ₃), 1.16 (d, J=6.4Hz, 2S C₄—CH ₃), 1.12 (d, J=7.5 Hz, 2R C₄—CH ₃), 1.08 (t, J=7.5 Hz, 2RH₇), 1.01 (t, J=7.5 Hz, 2S H₇). ¹³C NMR (226 MHz, CDCl₃, only the 2Repimer was detected): δ 205.59 (C₃), 170.21 (C₁), 78.68 (C₅), 50.56(C₂), 44.52 (C₄), 24.19 (C₆), 10.09 (C₇), 9.90 (C₄—CH₃), 8.40 (C₂—CH₃).HR-ESI-MS [M−H]⁻: calculated for C₉H₁₃O₃, m/z 169.0870. found m/z169.0871.

The enzymatic F-TKL was compared against an authentic synthetic standardby LC-MS, HR-ESI-MS, ¹⁹F-NMR, and GC-MS (FIG. 4, FIG. 17). ¹⁹F-NMR (565MHz, CDCl₃, CFCl₃=0 ppm): −171.95 (broad singlet), −210.32 (d, J=45.8Hz). GC-MS: t_(R), 8.56 min; EI spectrum (FIG. 17). HR-ESI-MS [M−H]⁻:calculated for C₈H₁₀FO₃, m/z 173.0619. found m/z 173.0623.

Enzymatic Preparation of F-TKL from Fluoroacetate

One-pot reaction mixtures containing 200 mM HEPES, pH 7.5, TCEP (2 mM),bovine serum albumin (3 mg/mL), magnesium chloride (5 mM), fluoroacetate(10 mM), NDK-SNAC (10 mM), coenzyme A (2 mM), sodium bicarbonate (75mM), ATP (2.5 mM), phosphoenolpyruvate (50 mM), pyruvate kinase (18U/mL), myokinase (10 U/mL), AckA (10 μM), Pta (1 μM), ACCase (15 μM),MatB (40 μM), methylmalonyl-CoA epimerase (5 μM) and DEBS_(Mod6)+TE (10μM) in a total volume of 1000 μL were incubated at 37° C. for 1.5 hrs atwhich time sodium phosphate pH 7.5 (400 mM) was added to the reaction.The reaction was incubated at 37° C. for a further 24 hrs. An aliquot(200 μL) was removed and prior to analysis, the aliquot was quenched bythe addition of 70% perchloric acid (10 μL). F-TKL production wasanalyzed by LC-MS as described above using single ion monitoring at m/z173 in negative ion mode. Telescope reaction mixtures containing 200 mMHEPES, pH 7.5, TCEP (2 mM), bovine serum albumin (3 mg/mL), magnesiumchloride (5 mM), fluoroacetate (10 mM), coenzyme A (1 mM), sodiumbicarbonate (75 mM), ATP (2.5 mM), phosphoenolpyruvate (50 mM), pyruvatekinase (18 U/mL), AckA (10 μM), Pta (1 μM) and ACCase (15 μM) in a totalvolume of 1000 μL were incubated at 37° C. for 1.5 hrs. The reaction wasthen spun through an Amicon spin concentrator (MWCO 3 kD) to removeproteins. 792 μL of the flow through was used to prepare a reaction with400 mM sodium phosphate, pH 7.5, TCEP (2 mM), magnesium chloride (10mM), NDK-SNAC (10 mM), phosphoenolpyruvate (50 mM), pyruvate kinase(18U/mL), myokinase (10 U/mL), MatB (40 μM), methylmalonyl-CoA epimerase(5 μM) and DEBS_(Mod6)+TE (10 μM) in a total volume of 1000 μL. Thereactions were allowed to proceed for 24 h and were assayed as describedfor the one-pot reactions.

(2S,3R)-1-((S)-4-Benzyl-2-oxooxazolidin-3-yl)-2-methyl-1-oxopentan-3-yl2-fluoroacetate(1)

(S)-4-benzyl-3-((2S,3R)-3-hydroxy-2-methylpentanoyl)oxazolidin-2-one(171 mg, 0.585 mmol) was prepared as previously described (Cane, D. E.,et al., J. Am. Chem., 115:527-535 (1993)) and combined with sodiumfluoroacetate (70 mg, 0.703 mmol, 1.2 eq) and HATU (267 mg, 0.702 mmol,1.2 eq) in a flame-dried round-bottom flask under a nitrogen atmosphere.Anhydrous THF (5.9 mL) and diisopropylethylamine (306 μL, 1.76 mmol, 3eq) were added and the reaction was capped and stirred vigorously atroom temperature for 44 h, during which time the white suspension turnedorange-brown. The mixture was diluted with ethyl acetate and washed withsaturated sodium bicarbonate, resulting in two clear layers. Theorange-brown organic layer was washed again with saturated sodiumbicarbonate, dried over MgSO₄, filtered through a plug of silica, andconcentrated to give an orange-brown oil. The residue was purified byflash chromatography on silica (30 g) using a step gradient from 100%heptane to 25% ethyl acetate in heptane with the desired compoundbeginning to elute in 20% ethyl acetate. Fractions were concentrated toyield the product (173 mg, 84%) as a clear, colorless oil, R_(f) 0.35(25% ethyl acetate/hexanes). ¹H NMR (500 MHz, CDCl₃): δ 7.39-7.16 (m,Ph-H), 5.31 (ddd, J=7.9, 5.9, 3.2 Hz, H₃), 4.87 (d, J=47.0, CH ₂F—C═O),4.60 (dddd, J=9.8, 7.7, 3.5, 2.3 Hz, H₄), 4.31 (ddd, J=8.7, 7.7, 0.8 Hz,H₅ pro-R), 4.19 (dd, J=8.9, 2.3 Hz, H₅ pro-S), 4.09 (qd, J=6.9, 3.2 Hz,H_(2′)), 3.28 (dd, J=13.4, 3.5 Hz, H_(6a)), 2.78 (dd, J=13.4, 9.8 Hz,H_(6b)), 1.79-1.64 (m, H_(4′)), 1.22 (d, J=6.9 Hz, C₂—CH ₃), 0.95 (t,J=7.4 Hz, H₅). ¹³C NMR (151 MHz, CDCl₃): δ 173.95 (C_(1′)), 168.04 (d,J=22.0 Hz, CH₂F—C═O), 153.83 (N—C═O—O), 135.41 (C_(aryl)), 129.56(C_(aryl)), 129.07 (C_(aryl)), 127.48 (C_(aryl)), 77.44 (d, J=182.3 Hz,CH₂F—C═O), 76.40 (C_(3′)), 66.58 (C₅), 55.94 (C₄), 41.00 (C_(2′)), 38.05(C₆), 25.16 (C_(4′)), 10.15 (C₂—CH₃), 10.04 (C_(5′)). ¹⁹F NMR (565 MHz,CDCl₃, CFCl₃=0 ppm): δ −230.44 (t, J=47.0 Hz). HR-ESI-MS [M+Na]⁺:calculated for C₁₈H₂₂FNO₅Na, m/z 374.1374. found m/z 374.1381.

Preparation of (2S,4S,5R)-2-fluoro-4-methyl-3-oxo-5-hydroxy-n-heptanoicacid 6-lactone (F-TKL)

Lactonization of 1 was carried out using literature methods (Hinterding,K., et al., Tetrahedron lett., 42:8463-8465 (2001)). 1 (160 mg, 0.455mmol) was dried under vacuum in a pear-shaped flask then placed undernitrogen. In a flame-dried round-bottom flask, anhydrous THF (4.5 mL)and LiHMDS (1.0 M in THF, 1.366 mL, 3 eq) were combined, stirred andcooled to −78° C. under nitrogen. The starting material was dissolved inanhydrous THF (3.5 mL), cooled to −78° C., and cannulated dropwise intothe solution of base over 20 min. A rinse of anhydrous THF (1.5 mL) wasalso transferred by cannula. The reaction mixture was stirred for 3 h at−78° C. and quenched by addition of saturated ammoniumchloride/methanol/water (1:1:1 v/v/v, 13 mL). The mixture was thenallowed to warm to room temperature while stirring. The pH of thequenched mixture was adjusted to 9 using 10 M NaOH and extracted with3×40 mL ethyl acetate to remove the oxazolidinone auxiliary. The aqueouslayer was adjusted to pH 2 using 12 M HCl and then extracted with 5×20mL dichloromethane. The combined organic layers were concentrated togive a clear, colorless oil, which contained approximately 1 mol %starting material by ¹H NMR (36 mg, 45%). The product was furtherpurified by flash chromatography on silica by washing extensively withdichloromethane (R_(f)<0.05) then eluting with ethyl acetate(R_(f)˜0.4), and concentrated to yield a white, crystalline solid (13mg, 16%). A mixture of enol (53%) and keto (47%) tautomers was observedin CDCl₃ (FIGS. 14 and 15). The keto form was almost exclusively the 2Sdiastereomer as determined by ¹H NOESY and molecular modeling (FIG. 16).The doublet in the ¹⁹F NMR spectrum in CDCl₃ at −205.96 ppm was assignedto the 2R keto diastereomer based on ¹H-¹⁹F HMBC (FIG. 16C). ¹H NMR (500MHz, CDCl₃): δ 5.83 (d, J=45.7 Hz, keto H₂), 4.71 (ddd, J=8.4, 5.2, 3.0Hz, keto H₅), 4.26 (ddd, J=8.8, 6.0, 3.3 Hz, enol H₅), 2.68 (qd, J=7.5,3.0 Hz, keto H₄), 2.45 (qt, J=7.2, 3.8 Hz, enol H₄), 1.88-1.78 (m, ketoH_(6a)), 1.75 (m, enolH_(6a)), 1.60 (m, keto H_(6b)), 1.55-1.44 (m,enolH_(6b)), 1.14 (d, J=7.5 Hz, keto H₈), 1.11 (d, J=7.1 Hz, enol H₅),1.00 (t, J=7.4 Hz, keto H₇), 0.92 (t, J=7.5 Hz, enol H₇). ¹³C NMR (151MHz, CDCl₃): δ 198.58 (d, J=13.4 Hz, keto C₃), 164.15 (d, J=20.0 Hz,keto C₁), 162.67 (d, J=24 Hz, enol C₁), 156.94 (d, J=6.5 Hz, enol C₃),130.00 (d, J=232.3 Hz, enol C₂), 89.82 (d, J=206.9 Hz, keto C₂), 80.27(enol C₅), 77.89 (d, J=1.7 Hz, keto C₅), 43.97 (keto C4), 35.84 (enolC₄), 24.15 (keto C₆), 23.96 (enol C₆), 10.38 (d, J=2.7 Hz, enol C₈),10.11 (keto C₈), 9.93 (keto C₇), 9.72 (enol C₇). ¹⁹F NMR (565 MHz,CDCl₃, CFCl₃=0 ppm): δ −172.36 (d, J=4.3 Hz, enol), −205.96 (d, J=44.9Hz, 2S keto), −210.40 (d, J=45.6 Hz, 2R keto). ¹⁹F NMR (565 MHz, 10%D₂O, 50 mM sodium phosphate pH 4.5): δ −178.66. ¹⁹F NMR (565 MHz, 15%D₂O, 85 mM Tris pH 7.5, 5-fluorouracil=−168.3 ppm): δ −190.20. GC-MS:t_(R), 8.51 min; EI spectrum (FIG. 17). HR-ESI-MS [M−H]⁻: calculated forC₈H₁₀FO₃, m/z 173.0619. found m/z 173.0617.

GC-MS Analysis of F-TKL

Samples were dissolved in dichloromethane and BSTFA containing 1%trimethylsilyl chloride (Sigma-Aldrich, 0.1 volumes) was added. Sampleswere analyzed on a Trace GC Ultra (Thermo Scientific) coupled to a DSQIIsingle-quadrupole mass spectrometer using an HP-5MS column (0.25 mm×30m, 0.25 μM film thickness, J & W Scientific). The injection volume was 1μL and the oven program was as follows: 75° C. for 3 min, ramp to 25° C.at 25° C. min⁻¹, ramp to 300° C. at 50° C. min⁻¹, hold for 1 min. Thecomparison between the synthetic and enzymatic F-TKL is shown in FIG.17.

Covalent Inhibition Assay for DEBS_(Mod6)+TE

Two triketide reaction mixtures (200 μL) were prepared as describedabove, one containing fluoromalonate (10 mM) and the othermethylmalonate (10 mM). DEBS_(Mod6)+TE (10 μM) and NDK-SNAC (2.5 mM)were added to each and the reactions were incubated at 37° C. for 18 h.The protein fraction was isolated from each mixture at room temperatureby desalting on a Sephadex G-25 column (3 mL) using 400 mM sodiumphosphate, pH 7.5. Fractions were pooled by Bradford assay andconcentrated to 200 μL using Amicon Ultra spin concentrators (3 kDaMWCO). The isolated DEBS_(Mod6)+TE was assayed by adding TCEP (2.5 mM),methylmalonyl-CoA (1 mM) and NDK-SNAC (1 mM) to this mixture to give afinal volume of 210 μL and incubating at 37° C. for 3 h, then analyzedby HPLC as described above.

Triketide Lactone Production Using DEBS_(Mod3/6)+TE/AT⁰

All assay mixtures contained 400 mM sodium phosphate, pH 7.5,phosphoenolpyruvate (50 mM), TCEP (5 mM), magnesium chloride (10 mM),ATP (2.5 mM), pyruvate kinase (27 U/mL), myokinase (10 U/mL),methylmalonyl-CoA epimerase (5 μM), CoA (1 mM), MatB (40 μM), methyl- orfluoromalonate (20 mM) and NDK-SNAC (5 mM). When used, DszsAT (5 μM) wasalso added to the reaction mixture. Reactions were initiated by additionof the appropriate DEBS+TE construct (Mod6 or Mod6/AT⁰, 10 μM; Mod3 andMod3/AT⁰, 5 μM in reactions containing DszsAT and 8 μM otherwise) andincubated at 37° C. for 18-20 h. Aliquots were removed, quenched,processed and analyzed as described above.

Triketide Lactone Production Using DEBS_(Mod2)/AT⁰

All assay mixtures contained 400 mM sodium phosphate, pH 7.5,phosphoenolpyruvate (20 mM), TCEP (5 mM), magnesium chloride (5 mM), ATP(2.5 mM), pyruvate kinase (18 U/mL), myokinase (10 U/mL),methylmalonyl-CoA epimerase (5 μM), CoA (1 mM), MatB (20 μM), NDK-SNAC(500 μM) and either methylmalonate, fluoromalonate or malonate (5 mM) asappropriate. Reactions were initiated by addition of DEBS_(Mod2)/AT⁰ (10μM) and incubated at 37° C. overnight. Aliquots were removed, quenchedby addition of HCl to 1 M, then processed and analyzed as describedabove.

Tetraketide Lactone Production

All reactions contained 400 mM sodium phosphate (pH 7.5 for the2,4-dimethyl- and 2-fluoro-4-methyl-tetraketide lactone reactions and pH6 for the 2-methyl-4-fluoro-tetraketide lactone reaction), glycerol(20%), phosphoenolpyruvate (20 mM), TCEP (10 mM), magnesium chloride (5mM), ATP (2.5 mM), pyruvate kinase (18 U/mL), myokinase (10 U/mL),methylmalonyl-CoA epimerase (5 μM), MatB (20 μM), CoA (1 mM),methylmalonyl-CoA (100 μM), NDK-SNAC (1 mM) and reduced nicotinamideadenine dinucleotide phosphate (NADPH; 5 mM).

The reaction to produce 2,4-dimethyl-tetraketide lactone also containedmethylmalonate (5 mM), DEBS_(Mod2) (10 μM) and DEBS_(Mod3)+TE (2 μM).The reaction to produce 2-fluoro-4-methyl-tetraketide lactone alsocontained fluoromalonate (5 mM), DEBS_(Mod2) (10 μM), DEBS_(Mod3)/AT⁰ (2μM) and DzAT (2 μM). The reaction to produce2-methyl-4-fluoro-tetraketide lactone also contained fluoromalonate (5mM), DEBS_(Mod2)/AT⁰ (10 μM) and DEBS_(Mod3) (2 μM).

All reactions were initialized by the addition of DEBS_(Mod2) orDEBS_(Mod2)/AT⁰ and incubated at 37° C. overnight. Reactions were thensaturated with sodium chloride and the aqueous layer was acidified bythe addition of 0.1 volumes of 70% perchloric acid and extracted fourtimes into 2 volumes of chloroform. The chloroform layer wasconcentrated by vacuum centrifugation and the tetraketide lactones wereresuspended in water for analysis. Tetraketide lactones were analyzed byLC-MS using a Phenomenex Kinetex XB-C18 1.7 μm 150×2.1 mm column with amobile phase of ammonium acetate (50 mM) with a gradient from 0 to 60%acetonitrile over 15 min and detected on an Agilent single quadruplemass spectrometer in negative ion mode.

ESI-MS/MS Analysis of Tetraketide Lactones

MS/MS spectra were collected using an LTQ FT (Thermo Scientific).Negative ions were generated using ES and analyzed in linear ion trapmode. MS/MS spectra were collected with the following normalizedcollision energies: TKL, 26; F-TKL, 35; tetraketide lactones, 26.

Triketide Lactone Production Under Competitive Conditions

For reactions with substrate regeneration, assay mixtures contained 400mM sodium phosphate, pH 7.5, phosphoenolpyruvate (50 mM), TCEP (5 mM),magnesium chloride (10 mM), ATP (2.5 mM), pyruvate kinase (27 U/mL),myokinase (10 U/mL), methylmalonyl-CoA epimerase (5 μM), NDK-SNAC (15mM) and DEBS_(Mod6)+TE (10 μM). Reactions were initiated by simultaneousaddition of MatB (40 μM), fluoromalonyl-CoA (1 μM) and malonyl-CoA (1μM) and incubated at 37° C. for 18-20 h. Protein was removed byfiltration through a 3 kDa NWCO membrane at 14,000×g and the filtratewas analyzed by LC-MS. Standards of H-TKL and F-TKL were prepared usinga mock reaction mixture (acyl-CoAs replaced by CoAs and NDK-SNACreplaced by N-acetyl cysteamine) as diluent. TKLs were quantified usingsingle ion monitoring, H-TKL in positive mode and F-TKL in negativemode. For reactions without substrate regeneration, assay mixturescontained 400 mM sodium phosphate, pH 7.5, TCEP (5 mM),methylmalonyl-CoA epimerase (5 μM), NDK-SNAC (3 mM) and DEBS_(Mod6)+TE(10 μM). Reactions were initiated by adding fluoromalonyl-CoA (1 mM) andmalonyl-CoA (1 mM) simultaneously. The mixtures were incubated at 37° C.for 20 h, then quenched, processed and analyzed as described above.H-TKL was monitored using an Agilent 6130 MS operating in positive ionmode, and the limit of detection was verified by spiking samples to 50nM H-TKL using a standard solution.

¹⁹F-NMR Analysis of E. coli

LB (250 mL) containing kanamycin and chloramphenicol (50 μg/mL each) ina 1 L baffled shake flask was inoculated to OD₆₀₀=0.05 with an overnightLB culture of E. coli BAP1 freshly co-transformed withpET28a-His₆-MatB.SCo and pTRC33-NphT7-PhaB. Cells were grown, induced,washed and resuspended at OD₆₀₀ 90-110 as described above for F-TKLproduction in resting cells. To 850 μL of this cell suspensionfluoromalonate (43 mM) was added and the cells were incubated at 16° C.for 1 d. Cells were pelleted by centrifugation at 18,000×g and thesupernatant (650 μL) was removed. The volume of the supernatant wasadjusted to 850 μL by addition of D₂O (extracellular fraction). Cellswere resuspended in 605 μL potassium phosphate buffer, pH 7.4, andcentrifuged again. The supernatant was removed and the cells wereresuspended to give a final volume of 850 μL, 17% D₂O and 35 mM sodiumphosphate pH 7.5. Cells were lysed by sonication and insoluble materialremoved by centrifugation at 18,000×g for 20 min at room temperature.The supernatant was removed and acidified by addition of 0.025 volumes70% perchloric acid. Insoluble material was removed by centrifugation at18,000×g for 20 min and the supernatant was removed. The pH was adjustedto 7 using 10 M sodium hydroxide (intracellular fraction). CFCl₃ wasadded as a chemical shift reference to both the extracellular andintracellular fractions, which were analyzed by ¹⁹F NMR using the ERETICmethod.

F-TKL Production in E. coli Cell Lysates

TB (1 L) containing Cb and Km (50 μg/mL each) in a 2.8 L Fernbachbaffled shake flask was inoculated to OD₆₀₀=0.05 with an overnight TBculture of E. coli BAP1 freshly co-transformed withpET28a-His₆-MatB.SCo/pRSG54 or pET28a/pET16b as the empty vectorcontrol. The cultures were grown at 37° C. at 250 rpm to OD₆₀₀=0.6 to0.8 at which point cultures were cooled on ice for 20 min, followed byinduction of protein expression with IPTG (0.2 mM) and overnight growthat 16° C. Cell pellets were harvested by centrifugation at 9,800×g for 7min at 4° C. and stored at −80° C. Frozen cell pellets were thawed andresuspended at 5 mL/g cell paste with sodium phosphate (500 mM, pH 7.5)and lysed by passage through a French pressure cell at 14,000 psi. Thelysate was centrifuged at 15,300×g for 20 min at 4° C. to separate thesoluble and insoluble fractions. Assay mixtures (100 μL) containingsoluble cell lysate (77 μL), phosphoenolpyruvate (50 mM), TCEP (5 mM),magnesium chloride (10 mM), pyruvate kinase (27 U/mL), myokinase (10U/mL), methylmalonyl-CoA epimerase (5 μM), fluoromalonate (10 mM),coenzyme A (500 μM), ATP (2.5 mM), and NDK-SNAC (10 mM) were incubatedovernight at 37° C. Reactions were quenched by the addition of 70%perchloric acid (5 μL) and insoluble material was removed bycentrifugation. Production of F-TKL was analyzed by LC-MS as describedabove.

F-TKL Production in E. coli Growing and Resting Cell Culture

LB (50 mL) containing carbenicillin and kanamycin (50 μg/mL each) withor without spectinomycin (100 μg/mL) in a 250 mL baffled shake flask wasinoculated to OD₆₀₀=0.05 with an overnight LB culture of E. coli BAP1freshly co-transformed with pET28a-His₆-MatB.SCo and the appropriateDEBS+TE plasmid, with or without pCDFDuet-DszsAT.

For F-TKL production in LB, cultures were grown at 37° C. at 200 rpm toOD₆₀₀=0.4 at which point cultures were cooled on ice for 10 min,followed by induction of protein expression with IPTG (0.2 mM). Thecultures were grown at 30° C. for 2 h following induction, at which theculture (10 mL) was transferred to a 30 mL tube. Fluoromalonate (50 mMfinal concentration), diethylfluoromalonate (10 mM final concentration,added as a 1 M solution in DMSO) and either NDK-SNAC (stock, 100 mMsolution in 10% DMSO; final, 5 mM) or 10% DMSO were added. The cultureswere grown at 30° C. for 20 h. The culture supernatant was collected bycentrifugation at 18,000×g for 15 min and acidified by addition of HClto a final concentration of 1 M. The acidified supernatant was thenextracted with 5×3 volumes of dichloromethane and the combined organiclayers were concentrated to 5-10 mL by rotary evaporation. The residuewas transferred to a silanized glass vial (Sigmacote®, Sigma-Aldrich)and water (200 μL) was added. The dichloromethane was removed from thebiphasic mixture by rotary evaporation and the aqueous solution oftriketide was analyzed by LC-MS as described above.

For F-TKL production by resting cells, cultures were grown at 37° C. at200 rpm to OD₆₀₀=0.8-0.9, at which point cultures were cooled on ice for15 min, followed by induction of protein expression with IPTG (0.2 mM).The cultures were grown at 16° C. for 20-24 h following induction. Cellswere collected by slow centrifugation at 1,000×g for 15 min at 4° C. Thecells were washed once with 100 mM potassium phosphate, pH 7.4, thenresuspended in the same buffer at an OD₆₀₀ of 90-110. To 50 μL of thissuspension in a 0.6 mL tube, fluoromalonate (50 mM final concentration)and NDK-SNAC (stock, 100 mM solution in 10% DMSO; final, 5 mM) wereadded. The cell suspensions were incubated with shaking at 16° C. for 20h. The culture supernatant was collected by centrifugation at 18,000×gfor 15 min and analyzed by LC-MS as described above with no furtherconcentration. The identity of the F-TKL produced in vivo was alsoconfirmed by HR-ESI-MS. HR-ESI-MS [M−H]⁻: calculated for C₈H₁₀FO₃, m/z173.0619. found m/z 173.0619.

Example 2 Supplementary Results

TABLE 1(A) Strains and plasmids used for this study. (B) Oligonucleotides used for gene andplasmid construction. (C) The primer map for construction of the synthetic nphT7 gene isalso shown with non-coding portions in lowercase.A. Strains and plasmids Strain Genotype Source BL21(de3)F⁻ ompT gal dcm lon Novagen hsdS_(B)(r_(B) ⁻ m_(B) ⁻) A(DE3 [lacIlacUV5-T7 gene 1 ind1 sam7 nin5]) BAP1 F⁻ ompT gal dcm lonPfeifer, B.A., et al., Science hsdS_(B)(r_(B) ⁻ m_(B) ⁻) A(DE3 [lacI291:1790-1792 (2001) lacUV5-T7 gene 1 ind1 sam7 nin5]) ΔprpRBCDE(sfp (T7), prpE (T7))] Plasmid Description Source pET16b-His₁₀-NphT7His₁₀-nphT7 (T7), lacI, Cb^(r), This study ColE1 pET16b-His₁₀-AckA.ECHis₁₀-ackA.EC (T7), lacI, This study Cb^(r), ColE1 pET16b-His₁₀-Pta.ECHis₁₀-pta.EC (T7), lacI, Cb^(r), This study ColE1 pET28a-His₆-MatB.SCoHis₆-matB.SCo (T7), lacI, This study Km^(r), ColE1 pET28a-His₆-Epi.SCoHis₆-epi.SCo (T7), lacI, This study Km^(r), ColE1 pET16b-His₁₀-THNSHis₁₀-thns (T7), lacI, Cb^(r), This study ColE1 pCDFDuet-DszsAT.SCe (T7), matB.SCo This study DszsAT.SCe-MatB.SCo(T7), lacI, Sp^(r), CloDF13 pCDFDuet-ø-MatB.SComatB.SCo (T7), lacI, Sp^(r), This study CloDF13 pFW3DszsAT.SCe-His₆ (T7), lacI, Wong, F.T., et al., Biochemistry,Cb^(r), ColE1 49:95-102 (2009) pTRC33-NphT7-PhaBnphT7.phaB (trc), lacIq, This study Cb^(r), M13 pBP19DEBS_(Mod2)-His₆(T7), lacI, Tsuji, S.Y., et al., Biochemistry,Cb^(r), ColE1 40:2326-2331 (2001) pSV272- His₆-MBP-His₆-MBP-DEBS_(Mod2) (T7), This study DEBS_(Mod2) lacI, Km^(r), ColE1pSV272-His₆-MBP- His₆-MBP-DEBS_(Mod2)/AT⁰ This study DEBS_(Mod2)/AT⁰(T7), lacI, Km^(r), ColE1 pAYC138 DEBS_(Mod6) + TE/AT⁰-His₆Wong, F.T., et al., Biochemistry, (T7), lacI, Cb^(r), ColE149:95-102 (2009) pRSG54 DEBS_(Mod6) + TE-His₆(T7),Gokhale, R.S., et al., Science, lacI, Cb^(r), ColE1 284:482-485 (1999)pRSG34 DEBS_(Mod3) + TE-His₆(T7), Gokhale, R.S., et al., Science,lacI, Cb^(r), ColE1 284:482-485 (1999) pAYC136 DEBS_(Mod3) + TE/AT⁰-His₆Wong, F.T., et al., Biochemistry, (T7), lacI, Cb^(r), ColE149:95-102 (2009) pRARE2 ileX, argU, thrU, tyrU, glyT, NovagenthrT, argW, metT, leuW, proL, lad, Cm^(r), p15a pBAD33-BirAbirA.EC (ara), araC, Cm^(r), This study p15aB. Oligonucleotide sequences Name Sequence nphT7 R1aaacgaacgtcggtcatggtg nphT7 F1 caccatgaccgacgttcgttttcgtatcattggcacgggtnphT7 R2 gctccggcacgtacgcacccgtgccaatgatacga nphT7 F2gcgtacgtgccggagcgtattgtgtccaacgacgaggt nphT7 R3accagccggcgcacccacctcgtcgttggacacaatac nphT7 F3gggtgcgccggctggtgttgatgatgactggattacccgt nphT7 R4cgttgacgaatgccggtcttacgggtaatccagtcatcatcaac nphT7 F4aagaccggcattcgtcaacgtcgttgggcggcggac nphT7 R5tcggaggtcgcttggtcgtccgccgcccaacga nphT7 F5gaccaagcgacctccgacctggcaaccgcggcg nphT7 R6tcaacgccgcacgacccgccgcggttgccagg nphT7 F6ggtcgtgcggcgttgaaagcagcgggtattacgcc nphT7 R7gcaataaccgtcagttgctccggcgtaatacccgctgctt nphT7 F7ggagcaactgacggttattgcggtcgcaacgtccaccc nphT7 R8ggctgcggacggtccggggtggacgttgcgacc nphT7 F8cggaccgtccgcagccgccgacggcggcctac nphT7 R9cgcccagatgatgttgcacgtaggccgccgtcggc nphT7 F9gtgcaacatcatctgggcgcaaccggcaccgcggc nphT7 R10tgcacacagcgttaacatcaaatgccgcggtgccggttg nphT7 F10atttgatgttaacgctgtgtgcagcggcacggtttttgct nphT7 R11ccgccacgctggacagagcaaaaaccgtgccgc nphT7 F11ctgtccagcgtggcgggcacgctggtgtatcgtgg nphT7 R12caatgaccagtgcgtaaccgccacgatacaccagcgtgc nphT7 F12cggttacgcactggtcattggtgccgatctgtattcccgta nphT7 R13ggtccgccggattcagaatacgggaatacagatcggcac nphT7 F13ttctgaatccggcggaccgcaagaccgttgttctgtttgg nphT7 R14cgcacccgcgccgtcaccaaacagaacaacggtcttgc nphT7 F14tgacggcgcgggtgcgatggtgctgggtccgac nphT7 R15acccgtacccgtgctggtcggacccagcaccat nphT7 F15cagcacgggtacgggtccgatcgtccgtcgcg nphT7 R16caaacgtgtgcagggcaacgcgacggacgatcgg nphT7 F16ttgccctgcacacgtttggtggtctgaccgacctgatt nphT7 R17cacccgccggcacacgaatcaggtcggtcagaccac nphT7 F17cgtgtgccggcgggtggcagccgccaaccgct nphT7 R18tccaagccatccgtgtccagcggttggcggctgc nphT7 F18ggacacggatggcttggacgcgggtctgcaatacttcg nphT7 R19cctcgcgaccgtccatagcgaagtattgcagacccgcg nphT7 F19ctatggacggtcgcgaggtgcgtcgttttgttaccgaac nphT7 R20cctttaatcagttgcggcaagtgttcggtaacaaaacgacgca nphT7 F20acttgccgcaactgattaaaggtttcttgcacgaggcggg nphT7 R21gctaatatctgccgcatcgacacccgcctcgtgcaagaaa nphT7 F21tgtcgatgcggcagatattagccattttgtgccgcaccaagc nphT7 R22cgtccagcatgacaccgttcgcttggtgcggcacaaaatg nphT7 F22gaacggtgtcatgctggacgaggtctttggtgaactgcacc nphT7 R23atggtcgcacgcggcaggtgcagttcaccaaagacct nphT7 F23tgccgcgtgcgaccatgcaccgtaccgtcgaaacc nphT7 R24cgcacccgtattgccgtaggtttcgacggtacggtgc nphT7 F24tacggcaatacgggtgcggccagcattccgattacgatg nphT7 R25tgcacggactgctgcatccatcgtaatcggaatgctggc nphT7 F25gatgcagcagtccgtgcaggtagcttccgtccggg nphT7 R26gccagcaggaccagttcacccggacggaagctacc nphT7 F26tgaactggtcctgctggcgggttttggtggtggcatg nphT7 R27gcgcgaagctcgctgccatgccaccaccaaaaccc nphT7 F27gcagcgagcttcgcgctgatcgagtggtaagtcagcc nphT7 R28acccgctctagccgtcaggctgacttaccactcgatca nphT7 F28 tgacggctagagcgggtNphT7 G F aatttcacacgagctcggtacccgggaggagatataccatgaccgacgttcgttttcgNphT7 G R gcgctgggtcattatatatctccttttcttaccactcgatcagcgcgaag PhaB Fgaaaaggagatatataatgacccagcgcatcgcttacgtaacc PhaB Rgcttgcatgcctgcaggtcgactctagaggatctcatgccttggctttgacgtatc MatB.SCo Ftcgattgcacatatgtcctctctcttcccggccctct MatB.SCo Ratcggatagctcgagtcagtcacggttcagcgcccgctt Epi.SCo Fatcccgaatcatatgctgacgcgaatcgacca Epi.SCo Rttagtctggctcgagtcagtgctcaggtgactcaa AckA.EC Fggagatatacatatgtcgagtaagttag AckA.EC R attggatcctctagatcaggcagtcaggcgPta.EC F attcatatgtcccgtattattatgctgatc Pta.EC Rattctcgaggagggtaccgacgtcttac THNS.SCo Fattcatatggcgactttgtgcagaccctcggtgtccgtcccggagc THNS.SCo Rattactagttcatgcctgcctcaccctccgcgacacgccccgtg pCDF-MatB.SCo Fttagttaagtataagaaggagatatacatatgtcctctctcttcccggccctct pCDF-MatB.SCo Rgtttctttaccagactcgagggtacctcagtcacggttcagcgcccg pCDF-DszsAT.SCe Fgtttaactttaataaggagatataccatgaaagcatacatgtttcccgggc pCDF-DszsAT.SCe Rcttaagcattatgcggccgcaagcttgttacgacgacgaggggctggg MBP-M2 Fgggatcgaggaaaacctgtattttcagggcatgagcggtgacaacggcatgaccgagg MBP-M2 RgcttgtcgacggagctcgaattcggggatcctcagtggtggtggtggtggtgctcgagtgMBP-M2ATnull FgttatcggtcacgcgcagggtgaaatcgcggccgcggtggtggcgggagcgttgtcgctgMBP-M2ATnull RcgcgatttcaccctgcgcgtgaccgataacggccgaaggaacggcaccgcaggcacgccaC. Primer map for synthetic nphT7 construction

GTTGGGCGGC

CAACCCGCCG

CGTGCGGCGT

TGCACGTTGT

GCTGTGTGCA

CATAAGACTT

GCGGGTGCGA

ACGGCCGCCC

GGTCTGCAAT

CCAGACGTTA

TACGCCGTCT

CTGGACGAGG

ACCTACGTCG

CTGGCGGGTT

TABLE 2 Rates of acyl-CoA hydrolysis by DEBS_(Mod6) + TE. Steady-statehydrolysis rates were measured using DEBS_(Mod6) + TE (1 μM) andacyl-CoA (500 μM). Values are reported as the mean ± s.d. (n = 4). v₀(μM min⁻¹) Relative rate Methylmalonyl-CoA 1.36 ± 0.05 1.0Fluoromalonyl-CoA 3.5 ± 0.3 2.6 Malonyl-CoA 6.1 ± 0.4 4.5

TABLE 3 F-TKL and H-TKL production under competitive conditions.DEBS_(Mod6) + TE was incubated with equimolar amounts of malonyl-CoA andfluoromalonyl-CoA (1 mM). Without substrate regeneration, no detectableH-TKL was formed (<50 nM). MatB and regeneration enzymes were thenincluded to amplify and quantify H-TKL formation. Values are reported asthe mean ± s.d. (n = 3). [F-TKL]/ Condition [F-TKL] (nM) [H-TKL] (nM)[H-TKL] No regeneration 450 ± 60 <50 >9 MatB regeneration 7,390 ± 520 720 ± 50 10.3 ± 0.1

TABLE 4 Concentrations of extra- and intracellular organofluorines influorohydroxybutyrate-producing cells. Fluoromalonate (42 mM) was addedto a suspension of cells expressing MatB, NphT7, and PhaB.Organofluorine concentrations were determined by ¹⁹F NMR using theERETIC method and normalized to the total suspension volume, of whichthe wet cell pellet constituted 33%. Chemical shifts are reportedrelative to the internal standard (CFCl₃). Concentration (mM) Species ∂(ppm) Extracellular Intracellular Fluoromalonate −178.3 36.9 0.9Fluorohydroxybutyrate −197.4 0.2 0.1 Fluoroacetate −217.9 1.1 0.2

Results and Discussion

The introduction of fluorine via synthetic or semisynthetic routes hasenabled the improvement of the clinical properties of several naturalproducts but remains challenging to achieve (Rivkin, et al., Org. Lett.,4:4081-4084 (2002); Bégué, et al., J. Fluorine Chem., 127:992-1012(2006); Llano-Sotelo, et al., Antimicrob. Agents Chemother, 54:4961-4970(2010); and Mo, et al., J. Am. Chem. Soc., 133:976-985 (2010)). Whileprevious studies have shown that distal fluorine substituents can beaccommodated in natural product biosynthetic pathways (Runguphan, etal., Proc. Natl. Acad. Sci. U.S.A., 106:13673-13678 (2009); and Goss, etal., ChemBioChem, 11:698-702 (2010)), access to fluoromalonyl-CoA, afluorinated analog of one of nature's most powerful carbon nucleophiles,as an extender unit would enable a general method for directincorporation of fluorine into any polyketide structure.

Many acetate-based natural products, polyketides in particular, aregenerated through the iterative condensation of activated thioesters,resulting in reactive β-keto units that condense further to produce awide range of structures (Staunton, et al., Nat. Prod. Rep. 18, 380-416(2001); Croteau, et al., in Biochemistry and molecular biology ofplants, R. B. Buchanan, W. Gruissem, R. Jones, Eds. (ASPB, Rockville,Md., 2000) pp. 1250-1318)). (FIG. 1B). The structural diversity ofpolyketides is especially striking given that the majority ofpolyketides draw on only two monomers, acetate and propionate, as theextender units that form their carbon skeletons (Cane, et al., Science,282:63-68 (1998); Staunton, et al., Nat. Prod. Rep., 18:380-416 (2001);and Chan, et al., Nat. Prod. Rep., 26:90-114 (2009)). Althoughpolyketide synthases (PKSs) have been observed to be promiscuous withregard to their starter units (McDaniel et al., Proc. Natl. Acad. Sci.U.S.A., 96:1846-1851 (1999)), the encoding of extender units has beenfound to be quite selective and many cellular acyl-CoAs are excludedfrom the backbone (Chan, et al., Nat. Prod. Rep., 26:90-114 (2009)).However, progress in engineering extender unit incorporation has beenmade by domain engineering (McDaniel, et al., Proc. Natl. Acad. Sci.U.S.A., 96:1846-1851 (1999); Sundermann, et al., ACS Chem. Biol.,8:443-450 (2012); and Koryakina, et al., Org. Biomol. Chem., 4449-4458(2013)) or incorporation via a domain that encodes a rare extender unit(Mo, et al., J. Am. Chem. Soc., 133:976-985 (2010); and Eustaquio, etal., J. Nat. Prod., 73:378-382 (2010)). Although fluoroacetate serves asa starter unit in nature to produce highly toxic ω-fluorofatty acids(FIG. 1A) (D. O'Hagan, J. Fluorine Chem., 127:1479-1483 (2006)),fluorine has never been observed to date within the backbone, implyingthat chain extension reactions with the fluorinated acyl-CoA do notoccur in these systems. The apparent inability of living systems toutilize fluoroacetate for the biosynthesis of complex small moleculeslikely results in part from the extreme properties of fluorine thataffect biological as well as chemical synthesis. For example, the pKa ofthe α-proton, electrophilicity of the carbonyl group, and the stabilityof the acyl-CoA and its corresponding carbanion are all highly impactedby fluorine substitution. Furthermore, the fluoroacetyl group bears aclear similarity to the fluoromethylketone motif used for the design ofcovalent inhibitors, suggesting that the irreversible alkylation ofactive-site nucleophiles could also create problems (Powers, et al.,Chem. Rev., 102:4639-4750 (2002)). Thus, the development of a system toincorporate fluorinated extender units could dramatically increase therange of complex structures that can be accessed but must also addressthe challenges involved in activating the fluoroacetate monomer for thedownstream CC bond forming chemistry involved in chain extensionreactions.

Chain elongation in polyketides and related fatty acid-based naturalproducts relies on a separate pool of extender units formed bycarboxylation of acyl-CoAs at the α-position. These malonyl-CoAderivatives are then used as masked enolates for CC bond formationfollowing decarboxylation. The fluorinated extender, fluoromalonyl-CoA,can be made through two routes: either a two-step activation of thebiogenic fluoroacetate or a direct ligation of CoA to fluoromalonate(FIG. 2). We reasoned that the acetate kinase (AckA)phosphotransacetylase (Pta) pair would be effective at fluoroacetateactivation, as mutations in this gene locus have been shown to lead tofluoroacetate resistance in Escherichia coli (Brown, et al., J. Gen.Microbiol., 102:327-336 (1977)). The enzymes from E. coli were thusoverexpressed and characterized biochemically, confirming that AckA andPta serve as an effective activation system to rapidly produce bothacetyl- and fluoroacetyl-CoA in nearly quantitative yield (FIGS. 5 and6). Analysis of the kinetic parameters for these enzymes with respect tofluorinated substrates indicated that neither appears to be affected bythe fluorine substituent beyond inductive effects that alter thenucleophilicity of the carboxylic acid (AckA) or electrophilicity of thecarbonyl (Pta). Next, we purified the individual AccABCD subunits thatmake up the acetyl-CoA carboxylase (ACCase) from E. coli and added theseenzymes to the AckAPta system in order to carry out the carboxylation offluoroacetate in a one-pot reaction to generate the fluoromalonyl-CoAextender unit (FIG. 2A, FIG. 5). Under these conditions, the ligation ofCoA with AckAPta to produce the acyl-CoA is rapid and production of thecarboxylated product is limited by the ACCase. Although the rate ofconversion is 4.5-fold slower for fluoroacetate compared to acetate, theoverall extent of reaction is similar for both congeners and suggeststhat covalent inactivation of the ACCase by fluoroacetyl-CoA is notsignificant. In addition to the route from fluoroacetate, we also testeda malonyl-CoA synthetase (MatB) (Hughes, et al., Chem. Biol., 18:165-176(2011)) for coupling CoA directly to fluoromalonate. Although MatBexhibits a 103-fold selectivity for malonate over fluoromalonate,fluoromalonyl-CoA is still produced at reasonable efficiency (FIG. 2B,FIGS. 7 and 8). Both of these systems also provide in situ regenerationcapacity that can amplify product yields from polyketide synthases andwe found that either system increased polyketide production bytetrahydroxynaphthalene synthase (Izumikawa, et al., J. Ind. Microbiol.Biot., 30:510-515 (2003)) compared to simple addition of malonyl-CoA(FIG. 9).

We next turned our attention to utilizing the fluoromalonyl-CoA monomerfor downstream chain elongation reactions. To start, we examined thebehavior of a simple polyketide synthase system with regard to one cycleof chain extension and ketoreduction, which is a key functionality oflarger multimodular systems for controlling downstream cyclization andrearrangements within the polyketide backbone (FIG. 3A) (Cane, et al.,Science, 282: 63-68 (1998); and Staunton, et al., Nat. Prod. Rep.,18:380-416 (2001)). We constructed a synthetic gene encoding NphT7 fromStreptomyces sp. CL190 (Okamura, et al., Proc. Natl. Acad. Sci. U.S.A,107:11265 (2010)), which appears to be a free-standing ketosynthase thatis related at the structural level to the ketosynthase domain of morecomplex polyketide synthases (FIG. 10), and isolated theheterologously-expressed enzyme for biochemical characterization (FIG.5). Using a coupled assay with an R-hydroxyl forming acetoacetyl-CoAreductase (PhaB), we found that NphT7 is competent to catalyze theformation of acetofluoroacetyl-CoA using an acetyl-CoA starter andfluoromalonyl-CoA extender with only a five-fold defect in catalyticefficiency (kcat/KM) derived from a drop in kcat with the fluorinatedsubstrate (FIG. 3). This lower turnover rate observed with thefluorinated substrate is possibly related to the reduced reactivity ofthe enolate species, which would be stabilized by the fluorinesubstituent. However, the overall yield was comparable for bothfluorinated and nonfluorinated substrates, which shows that adecarboxylative Claisen condensation with fluoromalonyl-CoA can takeplace at a similar extent of conversion compared to malonyl-CoA.

Furthermore, these experiments also show that the 2-fluoro-3-keto motifproduced with the fluoromalonyl-CoA extender can be accepted byketoreductases, as PhaB is capable of efficiently reducing theacetofluoroacetyl-CoA substrate (FIG. 11). The 1H and 19F NMR spectra ofthe reduced product indicate that both diastereomers are produced inthis reaction (FIG. 11), which may result either from lack ofstereochemical preference of NphT7 with respect to the fluorinesubstituent or from racemization of the product prior to reduction byPhaB. Although PhaB does not appear to show diastereoselectivity withrespect to the fluorine group, the polyketide synthase ketoreductasesare known to be selective with regard to their native α-substituent andcould potentially carry out the stereochemical resolution of thefluorine modification upon reduction (Siskos, et al., Chem. Biol.,12:1145-1153 (2005)).

With this information in hand, we sought to extend our biosyntheticmethod for fluorine introduction to more complex polyketide synthasesystems, which use the chain elongation reaction for the biosynthesis ofmany bioactive and clinically important natural products, such aserythromycin and rapamycin (Cane, et al., Science 282:63-68 (1998); andStaunton, et al., Nat. Prod. Rep. 18, 380-416 (2001)). Of themultimodular polyketide systems, 6-deoxyerythronolide B synthase (DEBS)is likely the best understood and also responsible for production of theerythromycin precursor (Khosla, et al., Annu. Rev. Biochem., 76:195-221(2007)). We therefore focused our studies on the sixth module of DEBS,including the terminal thioesterase (DEBSMod6+TE) (Gokhale, et al.,Science, 284:482-485 (1999)). Using a diketide substrate (NDK-SNAC),DEBSMod6+TE can catalyze a single round of chain elongation with itsnative methylmalonyl-CoA extender unit and then cyclize the tetheredproduct to form a methyltriketide lactone (TKL) (FIG. 4A, R═CH₃; FIG.4B, 1; FIG. 12) (Wu, et al., J. Am. Chem. Soc., 122:4847-4852 (2000)).We found that DEBSMod6+TE is also able to accept the fluorinated monomerin chain extension catalysis to form the 2-fluoro-2-desmethyltriketidelactone (F-TKL) and incorporate fluorine into the polyketide backbone(FIG. 4B, 2-4; FIG. 13). The identity of the F-TKL was established bycomparison to an authentic synthetic standard by reverse-phase HPLCmonitored by ESI-MS and further confirmed by characterization of theisolated compound by high resolution MS, GC-MS, and 19F NMR spectroscopy(FIGS. 14-17). Although the 2S keto tautomer is generated in ≧94%diastereomeric excess (d.e.) (FIG. 16), this ratio appears to be set bythe compound's stereoelectronic factors rather than the stereochemicalpreference of DEBSMod6+TE, as the FTKL is fully enolized in aqueoussolution. The F-TKL can also be produced directly from fluoroacetateusing the AckAPta/ACCase activation system in either a multi-stage (FIG.4B, 5-6) or single-pot reaction (FIG. 4B, 7-8) with DEBSMod6+TE in asimilar yield to the MatB reaction, which allows us to connectfluorinated polyketide production directly to the biosyntheticallyavailable fluorinated building block (FIG. 1A, Scheme 1).

In contrast to the chain extension reaction catalyzed by NphT7,DEBSMod6+TE does not incorporate fluorinated extender units into thetriketide lactone product as efficiently as its native methylmalonyl-CoAextender. Preliminary studies indicate that the reduced efficiency ofDEBSMod6+TE with the fluorinated extender is not due to covalentinactivation of the enzyme (FIG. 18), but rather to the more complexbiochemistry of polyketide synthases with regard to monomer selection(Bonnett, et al., Chem. Biol., 18:1075-1081 (2011)). Extender unithydrolysis, which occurs even for the native substrate (Table S2),appears to limit fluoromalonyl-CoA incorporation based on theobservations that MatB and ATP are needed for fluoromalonyl-CoAregeneration and that fluoromalonate remains the major organofluorinespecies even in their presence (FIG. 19). The fluoromalonyl-CoA extenderis however incorporated at higher efficiency by DEBSMod6+TE thanmalonyl-CoA (R═H), which is reported to be naturally excluded by DEBS(Liou, et al., Biochemistry, 42:200-207 (2002)). In fact, DEBSMod6+TEproduces at least 10-fold more F-TKL than H-TKL in a direct competitionexperiment with equimolar amounts (1 mM) of fluoromalonyl-CoA andmalonyl-CoA (Table 3).

To address the issue of site- or regioselective fluorine incorporation,we turned our attention to exploiting the greater reactivity of thefluorinated extender unit towards acylation reactions. In this regard,we hypothesized that it would be possible for a fluorinated substrate toselectively acylate either the AT or ACP domains of individual DEBSmodules in the presence of a catalytically compromised or inactive ATdomain, an approach that has been shown to facilitate malonylincorporation by DEBS (Kumar, et al., J. Am. Chem. Soc., 125:14307-14312(2003)). Experiments with DEBSMod6+TE showed that not only does F-TKLyield increase as expected but fluorine selectivity also improves uponintroduction of a key S2107A mutation, reversing the selectivity of thewild-type module (FIG. 4C).

Indeed, when the NDK-SNAC substrate is used with its native module,DEBSMod2, in conjunction with the analogous S2652A mutation, extensionwith fluoromalonyl-CoA to form FTKL reaches 30% efficiency compared tomethylmalonyl-CoA (FIG. 20). Furthermore, we found that the standalonetrans-AT from the disorazole polyketide synthase (Wong, et al.,Biochemistry, 49:95-102 (2009); and Wong, et al., Biochemistry,50:6539-6548 (2011)) accepts fluoromalonyl-CoA and can further enhanceF-TKL formation by the AT-null mutant (FIG. 4C). Using this approach, webegan to explore the possibility of site-selective fluorineincorporation with a mini-PKS model system, consisting of DEBSMod2 andDEBSMod3+TE, that was designed to carry out two chain extensionreactions from the NDK-SNAC substrate (Tsuji, et al., Biochemistry,40:2326-2331 (2001)).

Using the appropriate AT-null constructs, we were able to observeexclusive production of either regioisomer of the fluoro-methyltetraketide lactone (tetraKL). The identity of the 2-fluoro-4-methyltetraKL and 2-methyl-4-fluoro tetraKL were established by both HR ESI-MSand LC-MS based on their different retention times, as well as theirmass fragmentation patterns which are consistent with the incorporationof fluorine at the expected sites (FIG. 4D, FIG. 21). These studies alsoindicate that further chain extension after fluorine insertion can beachieved and that downstream reactions of fluorinated intermediatescould potentially be tolerated. This observation is consistent withprevious work that has shown that intermediates with non-nativesubstituents, including fluorine, can be extended and tailored to thefinal structure (Cane, et al., Science 282:63-68 (1998); Mo, et al., J.Am. Chem. Soc., 133:976-985 (2010); Runguphan, et al., Proc. Natl. Acad.Sci. U.S.A., 106:13673-13678 (2009); Goss, et al., ChemBioChem,11:698-702 (2010); Staunton, et al., Nat. Prod. Rep., 18:380-416 (2001);and McDaniel, et al., Proc. Natl. Acad. Sci. U.S.A., 96:1846-1851(1999)) and gives promise that larger fluorinated polyketide targets maybe accessible through this approach.

The observed selectivity for fluoromalonyl- over malonyl-CoA extenderunits suggested that polyketide chain extension reactions withfluoromalonyl-CoA could possibly be catalyzed in vivo in E. coli, whichcontains a significant malonyl-CoA pool (−35 μM) (Bennett, et al., Nat.Chem. Biol., 5:593-599 (2009)) but almost no methylmalonyl-CoA (Haller,et al., Biochemistry, 39:4622-4629 (2000); and Pfeifer, et al., Science,291:1790-1792 (2001)). We carried out preliminary 19F-NMR studies ofcells expressing MatB, NphT7 and PhaB and fed with non-toxic levels offluoromalonate. Analysis of the media and cell extracts indicated thatflux through fluoromalonyl-CoA could reach 100 μM to 1 mM, which issufficient for use by PKSs in live cells (Table S4). Next, we tested theability of DEBSMod6+TE to catalyze chain elongation in cell lysatesprepared from E. coli BAP1 coexpressing DEBSMod6+TE and MatB. Underthese conditions, F-TKL is produced with no observable H-TKL uponaddition of only NDK-SNAC, fluoromalonate, CoA, ATP, and the ATPregeneration system (FIG. 22A). Negative controls with either noDEBSMod6+TE/MatB expressed or no NDK-SNAC substrate show no productionof F-TKL (FIG. 22A). These results demonstrate that the intracellularlevel of expression of the DEBSMod6+TE and MatB enzymes is sufficientfor the incorporation of the fluorinated extender unit. They alsofurther imply that fluorine could be introduced into the polyketidebackbone inside living cells, which are capable of generating ATPthrough normal metabolic processes. We therefore cultured E. coli BAP1co-expressing DEBSMod6+TE and MatB and harvested the cells afterinduction. These cells were then fed with the fluoromalonate precursor,which resulted in the production of FTKL upon addition of NDK-SNAC (FIG.4B, 9; FIG. 22B). The identity of the F-TKL under these conditions wereestablished by LC-MS, co-injection with an authentic standard, as wellas high resolution MS. Moreover, F-TKL can also be produced directly incell culture with the simple addition of a mixture of both substrates tothe media after induction of DEBSMod6+TE and MatB (FIG. 22C). Takentogether, these studies show that the natural selectivity of thepolyketide synthase allows for the site-selective introduction offluorine over hydrogen into the polyketide backbone inside living cells.

Example 3

Preparation of chloromalonate. Diethylchloromalonate (Sigma-Aldrich; 0.5mL, 3.1 mmol) was saponified with methanolic sodium hydroxide (2 M, 3.5mL) in dichloromethane and methanol (9:1 v/v, 32 mL). The mixture wasconcentrated in vacuo and the residue dissolved in water, washed withdiethyl ether, then acidified to pH 2 with HCl and extracted with ethylacetate. The combined organic layers were dried (Mg5O4), filtered andconcentrated, and the residue dissolved in 1.5 M NaOH to give a 1 Msolution of sodium chloromalonate.

Chlorotriketide formation. The assay mixture contained 400 mM sodiumphosphate, pH 7.5, phosphoenolpyruvate (50 mM), TCEP (5 mM), magnesiumchloride (10 mM), ATP (2.5 mM), pyruvate kinase (27 U/mL), myokinase (10U/mL), CoA (1 mM), methylmalonyl-CoA epimerase (5 μM), MatB (40 μM) andchloromalonate (10 mM). This mixture was incubated at 37° C. for 45 minand then initiated by addition of the N-acetylcysteamine thioester of(2S,3R)-2-methyl-3-hydroxypentanoic acid (NDK-SNAC, 5 mM) andDEBSMod3+TE/AT0 (10 μM). Aliquots (35 μL) were removed and quenched byaddition of 70% perchloric acid (1.75 μL). Samples were centrifuged at18,000×g to pellet the precipitated protein. The supernatant (33 μL) wasremoved and excess salts were precipitated by freezing in liquidnitrogen and centrifuging at 18,000×g until thawed. The supernatant wasremoved and analyzed on a Zorbax Eclipse XDB C-18 column (3.5 μm, 3×150mm, 35° C., Agilent) using a linear gradient from 0 to 40% acetonitrileover 14 min with 0.1% formic acid as the aqueous mobile phase after aninitial hold at 0% acetonitrile for 30 s (0.8 mL/min) The product wasmonitored using an Agilent G1315D diode array detector (255 nm) and anAgilent 6130 single quadruple mass spectrometer operating in negativeion mode ([M−H]-m/z 189).

Published resources cited herein are incorporated by reference hereinfor their respective teachings of standard laboratory methods foundtherein. Such incorporation, at a minimum, is for the specific teachingand/or other purpose that may be noted when citing the reference herein.If a specific teaching and/or other purpose is not so noted, then thepublished resource is specifically incorporated for the teaching(s)indicated by one or more of the title, abstract, and/or summary of thereference. If no such specifically identified teaching and/or otherpurpose may be so relevant, then the published resource is incorporatedin order to more fully describe the state of the art to which thepresent invention pertains, and/or to provide such teachings as aregenerally known to those skilled in the art, as may be applicable.However, it is specifically stated that a citation of a publishedresource herein shall not be construed as an admission that such isprior art to the present invention. Also, in the event that one or moreof the incorporated published resources differs from or contradicts thisapplication, including but not limited to defined terms, term usage,described techniques, or the like, this application controls. Subjectmatter in the Examples is incorporated into this section to the extentnot already present.

While various embodiments of the present invention have been shown anddescribed herein, it is emphasized that such embodiments are provided byway of example only. Numerous variations, changes and substitutions maybe made without departing from the invention herein in its variousembodiments. Specifically, and for whatever reason, for any grouping ofcompounds, nucleic acid sequences, polypeptides including specificproteins including functional enzymes, metabolic pathway enzymes orintermediates, elements, or other compositions, or concentrations statedor otherwise presented herein in a list, table, or other grouping (suchas metabolic pathway enzymes shown in a figure), unless clearly statedotherwise, it is intended that each such grouping provides the basis forand serves to identify various subset embodiments, the subsetembodiments in their broadest scope comprising every subset of suchgrouping by exclusion of one or more members (or subsets) of therespective stated grouping. Moreover, when any range is describedherein, unless clearly stated otherwise, that range includes all valuestherein and all sub-ranges therein. Accordingly, it is intended that theinvention be limited only by the spirit and scope of appended claims,and of later claims, and of either such claims as they may be amendedduring prosecution.

What is claimed is:
 1. An enzymatically mediated method for producing ahalogenated ketide, said method comprising: (a) a first condensationreaction comprising contacting a polyketide synthase and a substrate forthe polyketide synthase with a compound according to Formula I:

 in which X is a member selected from F, Cl, Br and I, wherein saidpolyketide is competent to catalyze the condensation and the contactingis under conditions appropriate for the condensation.
 2. The methodaccording to claim 1, further comprising: (b) prior to (a), contacting aprecursor compound according to Formula II with one or more enzymecompetent to ligate Co-A onto a carboxyl moiety of the compoundaccording to Formula II under conditions appropriate for the ligation,thereby producing a compound according to Formula
 1. 3. The methodaccording to claim 1, wherein the halogenated ketide comprises at leastone subunit of a 2-halo-3-keto motif.
 4. The method according to claim2, wherein the at least one enzyme is a member selected from malonyl-CoAsythetase (MatB), acetate kinase, acyl-CoA synthase,phosphotransacetylase, acetyl coenzyme A carboxylase, and a combinationthereof.
 5. The method according to claim 1, wherein said polyketidesynthesase is selected from naturally occurring and non-naturallyoccurring polyketide synthases.
 6. The method of claim 1 wherein saidpolyketide synthase is deoxyerythronolide B synthase.
 7. The methodaccording to claim 1 wherein said polyketide synthase is expressedwithin a host microorganism, and said polyketide synthase is competentto perform the condensation within the host microorganism.
 8. The methodaccording to claim 1 wherein said polyketide synthase is expressedwithin a host microorganism, and said polyketide synthase is competentto perform the condensation within the host microorganism.
 9. The methodaccording to claim 7, wherein said host microorganism is fed a non-toxicamount of a compound according to Formula II.
 10. The method accordingto claim 7, wherein said host microorganism is competent to convert saidcompound according to Formula II into said compound according to FormulaI.
 11. The method according to claim 7, wherein said host microorganismis competent to convert said compound according to Formula III into saidcompound according to Formula II.
 12. The method according to claim 9,wherein said host microorganism expresses an enzyme selected fromacetate kinase, phosphotransacetylase, acetyl coenzyme A carboxylase anda combination thereof.
 13. The method according to claim 10, whereineach of said malonyl-CoA sythetase (MatB), acetate kinase,phosphotransacetylase, acyl-CoA synthase, acetyl coenzyme A carboxylaseis independently selected from naturally occurring and non-naturallyoccurring enzymes.
 14. The method according to claim 10, wherein saidacetyl coenzyme A carboxylase comprises a subunit selected from AccABCD,AccA2, PccBE and a combination thereof.
 15. The method according toclaim 1, further comprising carrying out a second condensationsubsequent to said first condensation reaction, wherein said halogenatedketide is said substrate.
 16. The method according to claim 3, whereinthe subunit of 2-halo-3-keto motif is reducible by a ketoreductase. 17.The method according to claim 1, further comprising, (c), following (a),dehydrating the product of step (a).
 18. The method according to claim17, further comprising (d), following (c), submitting the product of (c)to an enoyl reduction.
 19. The method according to claim 1, furthercomprising, prior to (a), transferring a halomalonyl subunit from acompound according to Formula I onto a polyketide synthase throughaction of trans-acyl transferase.
 20. A compound produced by a methodaccording to claim
 1. 21. A host microorganism cell comprising anon-toxic amount of a molecule selected from Formula I, Formula II,Formula III and a combination thereof within an internal space of thecell.